The A204 cell line, derived from a human rhabdomyosarcoma, was studied in culture for its capacity to synthesize collagen types and other extracellular matrix proteins. The cells synthesized and secreted into the culture medium collagenous proteins with apparent molecular weights of 220,000 and 150,000. These were identified as the pro alpha 1 and pro alpha 2 chains of type V collagen by immunoprecipitation and by peptide mapping. The pro alpha 1(V) chain was made in excess of a 2:1 ratio for pro alpha 1(V) to pro alpha 2(V), and a fraction of the pro alpha 1(V) chains together with all of the pro alpha 2(V) chains participated in intermolecular disulfide bonding. The chains were extensively glycosylated at hydroxylysyl residues in ascorbate-supplemented cultures. A fraction of the secreted pro alpha 1(V) chains was processed to the p alpha 1(V) form, but further processing in the culture medium was very slow and the type V collagen molecules deposits in the extracellular matrix apparently retained large non-triple helical domains. Since the A204 cell line does not produce other collagen types, it may prove useful in further studies of the biosynthesis of type V procollagen.