Extensive evidence demonstrates pronounced effects of relaxin on the differentiation of human endometrial cells in vitro. In vivo data in rhesus monkeys suggest a role for relaxin in the development of endometrial vascular architecture. In women, pregnancy can be established and maintained in the absence of circulating relaxin. Thus, local synthesis by the endometrium is necessary if relaxin plays a physiological role in human endometrial function. Although relaxin protein and the prorelaxin C peptide have been localized to human endometrium, no data for relaxin synthesis have been provided to date. We therefore assessed relaxin mRNA and protein levels in cultured, defined human endometrial cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques were used to demonstrate the presence of relaxin mRNA in human stromal and glandular epithelial cells. Secretion of the protein into the media of cultured cells of both types was also detected. Relaxin stimulated the expression of vascular endothelial growth factor in glandular epithelial and stromal cells that were isolated from tissue that had been taken during the secretory phase of the cycle. Relaxin inhibited the expression of procollagenase from both glandular epithelial cells, with a more marked inhibition demonstrated from cells that were isolated from tissue that had been taken during the secretory phase, and from stromal cells. These data demonstrate that human endometrial cells synthesize relaxin, and they support the concept that relaxin fosters endometrial conditions that are required for implantation in women.