Using [3H]thymidine incorporation into Balb/c 3T3 fibroblasts as an index of mitotic activity, several cationic growth-promoting peptide fractions of relatively low potency were identified and partially purified from bovine pituitaries. The most completely characterized fraction exhibited an isoelectric pH of 10.4, and gel exclusion chromatograms under non-denaturing conditions were consistent with a self-associating system. Gel exclusion chromatography in the presence of 6 M guanidine hydrochloride demonstrated that the major reduced and S-alkylated peptides comprising this fraction had molecular weights of 8400 and 9000, and there was no evidence of disulfides between these or other minor peptides. This was confirmed by polyacrylamide gel electrophoresis in the presence of sodium lauryl sulfate and urea. Two other active fractions, each with an isoelectric pH of 9.9, were found to have major reduced and S-alkylated peptides with molecular weights between 6400 and 13 000, and the chromatographic results suggested the presence of intramolecular disulfides. The peptides from all fractions had a relatively high percentage of basic amino acids (approx. 20%); lysine was predominant (10-17%) and histidine was relatively low (less than 1%). Based on various criteria including amino acid composition, molecular weight, isoelectric point, and biological assays, the mitogenic activity in these fractions does not appear to result from fibroblast growth factor. Thus, the pituitary seems to contain multiple cationic mitogenic peptides of low potency for fibroblasts in vitro and their target cells in vivo remain to be defined.