Biomedical techniques have been used to determine the submicrosomal localization of prolyl and lysyl hydroxylases, enzymes which catalyze the hydroxylation of the indicated amino acid residues in nascent procollagen chains. While 90-93% of prolyl hydroxylase was found to be intracisternal, only half of lysyl hydroxylase was. The other half was tightly bound to the microsomal membrane and required 0.1M NaCl plus detergent for release. The functional significance of such different distributions for two very similar enzymes is being investigated. Studies also have continued on the identification of a reducing cofactor for prolyl hydroxylase in Kirsten virus transformed Balb 3T3 cells (Ki-3T3). Ascorbic acid is not synthesized by these cells although proline hydroxylation is ascorbate-independent, but they do contain reduced pteridine which can act as a reductant for prolyl hydroxylase in vitro. There is approximately 7-fold more pteridine in transformed cells than in the parent which is ascorbate dependent, suggesting that a pteridine may be the active cofactor in Ki-3T3. Ki-3T3 has a relative rate of collagen synthesis about one-third that of the parent line and synthesizes a different genetic type of collagen than the parent, suggesting that viral transformation alters the genetic expression of collagen synthesis. Dibutyryl cAMP increases collagen synthesis in Ki-3T3 but does not cause reversion to the genetic pattern of the parent.