Long-term administration of prolactin (PRL) markedly enhanced the induction of epidermal squamous cell carcinomas by a chemical carcinogen, 3-methylcholanthrene [(MCA) CAS: 56-49-5], in Swiss male albino mice. DNA radioactivity and quantitative estimation of autoradiographs with the use of [3H]thymidine revealed a significant increase in DNA content (twofold) and in the percentage of labeled neoplastic nuclei in mice treated with PRL plus MCA (48.50%) as compared to that in mice treated with MCA alone (23.50%). Ultrastructural and cytologic studies revealed the predominance of a trabecular-hepatoid type of epidermal carcinoma with advanced nuclear irregularities, glycogen granules, mirror-image cells, and phagolysosomes in PRL-MCA-treated carcinomas as compared to characteristic squamous neoplastic cells with enlarged nuclei, tonofilaments, and keratin formation in epidermal carcinomas following treatment with MCA alone. Scanning electron microscopy revealed the predominance of rounded cells covered by numerous thick microvilli and blebs with an intense stromal reaction following PRL-MCA administration as compared to characteristic polyhedral cells covered by small microvilli following treatment with MCA alone. These findings demonstrate that PRL is an important hormone in epidermal neoplastic cell growth and differentiation.-JNCI 1985; 74:1335-1346.