Chlamydia trachomatisocular strains cause a blinding disease known as trachoma. These strains rarely cause urogenital infections and are not found in the upper genital tract or rectum. Urogenital strains are responsible for a self-limited conjunctivitis and the sequelae of infertility, ectopic pregnancy, and hemorrhagic proctitis. However, the differential cellular responses that drive these clinically observed disease outcomes are not completely understood. Primary conjunctival, endocervical, and endometrial epithelial and stromal fibroblast cells, HeLa229 cells, and immortalized conjunctival epithelial (HCjE) cells were infected with the ocular A/Har-13 (A) and Ba/Apache-2 (Ba) strains and urogenital D/UW-3 (D) and E/Bour (E) strains. Infection rates, progeny production, and cytokine/chemokine secretion levels were evaluated in comparison with those in uninfected cells. All strain types infected all cell types with similar levels of efficacy and development. However, progeny production levels differed among primary cells: Ba produced significantly more progeny than E in endocervical and endometrial fibroblasts, while A progeny were less abundant than E progeny.C.trachomatisinfection of primary epithelial cells elicited an increase in pro- and anti-inflammatory mediators compared to levels in uninfected cells, but there were no significant differences by strain type. In contrast, for primary fibroblasts, ocular strains elicited significant increases in the pro- and anti-inflammatory mediators macrophage inflammatory protein (MIP)-1β, thymus- and activation-regulated chemokine (TARC), interleukin (IL)-2, IL-12p70, and interferon gamma-induced protein 10 (IP-10) compared to levels in urogenital strains, while urogenital strains elicited a distinct and significant increase in the proinflammatory mediators IL-1α, IL-1β, IL-8, gamma interferon (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Our data indicate that primary fibroblasts, not epithelial cells, drive host inflammatory responses that are dependent on strain type and likely influence disease outcomes, establishing their importance as a novel model for studies ofC. trachomatisdisease pathogenesis.IMPORTANCEChlamydia trachomatisis a human pathogen and the leading cause of preventable blindness and sexually transmitted diseases in the world. CertainC. trachomatisstrains cause ocular disease, while others cause upper genital tract pathology. However, little is known about the cellular or immunologic basis for these differences. Here, we compared the abilities of the strain types to infect, replicate, and initiate an immune response in primary human ocular and urogenital epithelial cells, as well as in fibroblasts from the underlying stroma. While there were no significant differences in infection rates or intracellular growth for any strain in any cell type, proinflammatory responses were driven not by the epithelial cells but by fibroblasts and were distinct between ocular and urogenital strains. Our findings suggest that primary fibroblasts are a novel and more appropriate model for studies of immune responses that will expand our understanding of the differential pathological disease outcomes caused by variousC. trachomatisstrain types.