The most commonly identified mutations causing Ehlers-Danlos syndrome (EDS) classic type result in haploinsufficiency of pro␣1(V) chains of type V collagen, a quantitatively minor collagen that co-assembles with type I collagen as heterotypic fibrils. To determine the role(s) of type I/V collagen interactions in fibrillogenesis and elucidate the mechanism whereby half-reduction of type V collagen causes abnormal connective tissue biogenesis observed in EDS, we analyzed mice heterozygous for a targeted inactivating mutation in col5a1 that caused 50% reduction in col5a1 mRNA and collagen V. Comparable with EDS patients, they had decreased aortic stiffness and tensile strength and hyperextensible skin with decreased tensile strength of both normal and wounded skin. In dermis, 50% fewer fibrils were assembled with two subpopulations: relatively normal fibrils with periodic immunoreactivity for collagen V where type I/V interactions regulate nucleation of fibril assembly and abnormal fibrils, lacking collagen V, generated by unregulated sequestration of type I collagen. The presence of the aberrant fibril subpopulation disrupts the normal linear and lateral growth mediated by fibril fusion. Therefore, abnormal fibril nucleation and dysfunctional fibril growth with potential disruption of cell-directed fibril organization leads to the connective tissue dysfunction associated with EDS.Abnormal collagen fibril formation is a hallmark of several forms of the Ehlers-Danlos syndrome. The classic form of the Ehlers-Danlos syndrome (EDS 2 types I, II) is characterized by fragile, hyperextensible skin, widened, atrophic scars, bruisability, joint laxity, a high prevalence of aortic root dilation, and other manifestations of connective tissue weakness, including inguinal hernia and prolapse of the uterine cervix or rectum (1-3). The dermis of EDS patients contains large, very irregular collagen fibrils (4, 5). Heterozygosity for mutations that result in a reduced quantity or altered structure of type V collagen have been identified in up to one-half of all cases of the classic form of Ehlers-Danlos syndrome (EDS, types I/II) (6 -9, 11-15). The most common reported molecular mechanism in classic EDS, involving up to 40% of cases, is the functional loss of one allele of COL5A1, which encodes pro␣1(V) chains of type V collagen (16 -18).Type V collagen is a quantitatively minor fibril-forming collagen. Several isoforms of type V collagen exist, differing in the type and ratios of constituent ␣ chains, including heterotrimeric molecules containing type XI collagen chains. The pro␣1(V) chain, encoded by COL5A1 at human chromosomal locus 9q34, is the rate-limiting component of type V collagen trimer assembly by virtue of the eight-cysteine motif in the NC1 domain (19). The most abundant and most widely distributed isoform of type V collagen is the [␣1(V)] 2 ␣2(V)] heterotrimer that co-assembles with type I collagen as heterotypic fibrils (20). This isoform of type V collagen retains a non-collagenous, N-terminal domain that is presen...