A line of normal rat embryo fibroblasts was transformed with N-methyl-N’-nitro-N-nitrosoguanidine (a chemical carcinogen), SV40 and polyoma virus (two DMA viruses), and Rous sarcoma virus (an RNA tumor virus). In this study, we report a comparison of the levels of collagen synthesis and procollagen messenger RNA (mRNA) in 13 lines selected after transformation with one of these agents. Collagen synthesis and procollagen mRNA levels were compared with the degree of transformation determined from morphology, saturation density, growth in agarose, and tumorigenicity in nude mice. Each class of transformants had a characteristic level of collagen synthesis; this level correlated inversely with the degree of transformation of the rat embryo fibroblasts. In N-methyl-N’-nitro-N-nitrosoguanidine and SV40 transformants which were moderately transformed, collagen synthesis was hardly affected, but, in polyomavirus and Rous sarcoma virus transformants which were more severely transformed, collagen synthesis was 30 to 48% and 12 to 25%, respectively, of control levels. Type I procollagen mRNA activity measured in RNA from nine of the lines by an in vitro translation assay also decreased with increasing severity of transformation. Procollagen mRNA levels were reduced to about one-half of control levels in one SV40 transformant and to 17 to 23% of controls in polyoma virus and Rous sarcoma virus transformants. We conclude that, in this series of rat fibroblast lines, transformation with different agents resulted in characteristic levels of collagen synthesis and that collagen synthesis was most reduced in the cells which were most transformed by other criteria. © 1981, American Association for Cancer Research. All rights reserved.