Tissue homogenates of rat ventral prostate were examined for growth factor activity using a fibroblast mitogenesis assay. G-75 Sephadex gel filtration separated the growth factor activity into two peaks. A broad first peak contained 98% of the protein and several growth factor moieties. A smaller second peak (MW 6,000) contained epidermal growth factor (EGF) as determined by binding in both a competitive receptor binding assay and a radioimmunoassay using anti-mouse epidermal growth factor (anti-mEGF). The broad first peak also contained substantial amounts of EGF-like activity as higher MW forms of EGF. The broad first peak was further fractionated by heparin-Sepharose affinity chromatography. A major fraction with growth factor activity eluted at 1.5 M NaCl and this fraction was shown to contain bFGF by immunostaining with antisera prepared against synthetic peptides corresponding to amino acid sequences 1-12 (amino terminal), 33-43 (internal), and 136-145 (carboxy terminal) of basic fibroblast growth factor (bFGF). EGF-like and bFGF-like molecules account for the major growth factor activity in the rat ventral prostate.