Human myometrium is shown for the first time to produce prolactin in vitro. This prolactin is similar to pituitary prolactin by criteria of immunologic identity, gel chromatography and bioassay. The de novo synthesis of myometrical prolactin is supported by no detectable prolactin in initial tissue homogenate, nondetectable prolactin production during the first 24 hours of culture, cycloheximide inhibition of prolactin production with recovery of production in control medium, and tritiated leucine incorporation into prolactin. Although human myometrium is capable of producing prolactin without the addition of exogenous hormones, the addition of estrogen and progesterone, respectively, enhances and suppresses prolactin production in contrast to decidualized human endometrium where opposite effects on prolactin production are found.