The collagens synthesized by Chinese hamster ovary cells have been isolated and characterized. Although these cells produce very small amounts of collagen, at least five distinct collagenous chains could be identified from radiolabeled media and cell extracts after limited pepsin digestion. Two chains were characterized as alpha 1(V) and alpha 2(V), based on electrophoretic mobility, resistance to vertebrate collagenase, chromatographic properties on carboxymethylcellulose, and cyanogen bromide peptide patterns. Two smaller collagenous proteins (Mr 34000 and 37000) were also isolated by carboxymethylcellulose chromatography and characterized by cyanogen bromide digestion patterns. These collagens showed similarities to type IV collagen fragments but may be unique to Chinese hamster ovary cells. A colcemid-resistant mutant of Chinese hamster ovary cells designated CMR795 [Ling, V., Aubin, J.E., Chase, A., & Sarangi, F. (1979) Cell (Cambridge, Mass.) 18, 423-430] was found to synthesize the same collagen chains but in different proportions. In the wild-type cells colcemid (0.05-0.1 microgram/mL) reduced the amount of type V collagen in the culture media but had little effect on the other collagen type, whereas the type V collagen reduction was less pronounced in the CMR795 cells treated with the same concentrations of colcemid. Dibutyryladenosine cyclic monophosphate caused a fibroblast-like "reverse transformation" of the Chinese hamster ovary cells similar to that described previously [Hsie, A.W., & Puck, T. T. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 358-361]. However, collagen synthesis was increased only slightly. Furthermore, no apparent alteration in the types of collagens synthesized was detected.