In vivo and in-vitro endometrial stromal synthesis of prolactin occurs after progesterone-induced decidualization. Synthesis of prolactin by myometrium in vitro suggests that cells whose embryologic origin is the loose mesenchyme surrounding the paramesonephric ducts may retain the capacity to synthesize prolactin. Since physiologic myometrial synthesis of prolactin has not been demonstrated in vivo, prolactin genome expression in pathologic conditions was considered. Follicular phase leiomyomas were diced to 8 mm3 and cultured in Dulbecco''s modified Eagle''s medium (DMEM) with either no hormones, estradiol 200 pg/ml, progesterone 20 ng/ml, or estradiol and progesterone. Media were sampled and changed every other day for 8 days, followed by culture in 3H-labeled leucine DMEM for 2 days. Portions of leiomyomas were homogenized for initial prolactin content, and all samples were assayed for prolactin by radioimmunoassay. Follicular phase leiomyomas contained prolactin (47 .+-. 15 ng/g) in excess of normal serum values. Synthesis was demonstrated during all time periods from leiomyomas not exposed to progesterone. Progesterone variably suppressed the synthesis of prolactin until after 144 h of culture. Determination of MW on a 60 .times. 1.5 cm Sephadex G-100 column revealed identical estimates for pituitary, decidual, and leiomyoma prolactin. 3H-labeled leucine incorporation into prolactin was confirmed by immunoprecipitation of Sephadex G-100 column fractions. Similar antigenicity was confirmed by parallel dilution curves for pituitary, decidual, and leiomyoma prolactin. Preliminary bioactivity in lymphoma proliferation assays confirmed prolactin activity. Proliferative phase leiomyomas evidently contained elevated prolactin presumably secondary to in vivo synthesis. This synthesis was confirmed in vitro.