For the investigation of the pathogenesis of desmoplasia, the capacities to synthesize collagen in vitro of 2 bile duct carcinomas (lines 1 and 10) of Sewall-Wright inbred strain 2 guinea pigs and of syngeneic dermal fibroblasts were studied. line 10 cells synthesized collagen type IV as judged by sensitivity to bacterial collagenase, by immunoprecipitation, by migration of proα (IV) chains and pepsin-resistant fragments on sodium dodecyl sulfate-polyacrylamide gels, and by immunofluorescence. Line i cells also synthesized small amounts of collagenase-sensitive protein. Neither line 1 nor line 10 cells synthesized detectable collagen type I, III, or V. Only about 1% of [¹⁴C]proline incorporated by tumor cells was found in collagenase-sensitive protein. In contrast, dermal fibroblasts synthesized 4 and 128 times as much collagenase-sensitive protein as line 10 and line 1 cells, respectively, amounting to 20% of total protein synthesized. Fibroblasts produced mostly collagen types I and III, in a ratio of 7:1, and smaller amounts of collagen type V. Thus lines 1 and 10 carcinoma cells produce primarily basement membrane collagen, whereas interstitial collagens, abundant in desmoplastic tumor stroma, are fibroblast products.