The relaxin-like factor (RLF) was recently discovered as a new member of the insulin-insulin-like growth factor-relaxin family of growth factors and hormones predominantly in the Leydig cells of the testis. In cattle, in contrast to other species, the RLF gene is also expressed to a high level in the ovary, where its expression pattern in the corpus luteum of the late cycle and pregnancy is similar to that of relaxin in the pig. The RLF gene was also transcribed to a high level in the theca cells of estrogen-rich, large antral follicles. Long-term primary cultures of bovine theca cells showed that expression was insulin dependent. After an initial decline in specific mRNA concentrations, there was a switch to a transcript with a longer poly(A) tail at about Day 6 of culture, which continued to increase to very high levels by Day 15 of culture. Addition of fetal calf serum to cultures caused a rapid and irreversible down-regulation of the RLF gene. Also, LH caused a decline in specific gene expression in long-term primary theca cell cultures. As in the Leydig cells of the testis, the pattern of RLF gene expression appears to reflect the differentiation state of the ovarian theca-luteal cell lineage, and should prove useful for mapping the fate of these cells under differing stimulation regimes.