Background Cleaned electrocardiographic lead wires are a potential source of microorganisms capable of causing nosocomial infection. Objectives To examine fungal and bacterial growth on cleaned reusable lead wires, determine if microbial growth is associated with hospital site or work environment, determine the prevalence of antibiotic-resistant bacteria, and learn if antibioticresistant bacteria are associated with hospital site and work environment. Methods Cleaned lead wires (N = 320) from critical care and telemetry units, emergency departments, and operating rooms of 4 hospitals were swabbed and the specimens cultured for microbial growth. Bacterial species were grouped by their risk for human infection: at risk (n = 9), potential risk (n = 5), and no risk (n = 10). Work environments were compared by using pairwise contrasts from a generalized estimating equation model. Results Fungi were rare (0.6%). Of 226 cultures from 201 wires (62.8%) with bacterial growth, 121 were of at-or potential-risk bacteria (37.8%). Urban hospitals had less growth (P ≤ .001) and fewer bacterial species per wire (P ≤ .001) than did community hospitals. Presence of any bacteria (P = .02) and number of bacterial species per wire (P = .002) were lowest in operating rooms; emergency departments and telemetry units had more growth than did critical care units. Among specimens of staphylococci and enterococci, 6 each were sensitive to antibiotics; of 4 resistant staphylococcal species, 1 was not a human opportunistic pathogen and 3 were potential-risk species.