Relaxin has been demonstrated to be produced by the corpus luteum of the menstrual cycle and pregnancy and is also produced by the endometrium and decidua, although these nonluteal sources may not contribute to circulating relaxin concentrations. The reports of luteal production of relaxin have failed to consider nonluteal ovarian sources. To look for sources of nonluteal ovarian relaxin, human ovaries were collected from patients who underwent removal of the ovary for a variety of reasons. Tissues were fixed in formalin and embedded in paraffin. Two monoclonal antibodies were used for immunocytochemical staining, one directed against human relaxin and the other against the C peptide of prorelaxin. In addition to the expected staining of corpora lutea, the luteinized theca interna but not granulosa of developing follicles from ovaries with an active corpus luteum of the cycle also stained positive for both relaxin and prorelaxin. Ovaries from term pregnant women with luteinized theca also demonstrated staining for relaxin and prorelaxin. In addition to luteal and thecal cell staining, small clusters of pseudodecidual cells in the periphery of the ovary stained positive for relaxin and prorelaxin. These data indicate that the ovary contains theca interna-derived structural elements in addition to the corpus luteum that produce relaxin when a corpus luteum is active, while granulosa-derived elements do not. This suggests that luteal production of relaxin is from theca-derived elements and may explain instances of independent relaxin and progesterone secretion.