Previously, leptin has been found in human and rodent mammary tissue. The present research was conducted to determine (1) if leptin is produced by bovine mammary epithelial cells and (2) if leptin production in bovine mammary epithelial cells is hormonally regulated. Western blot analysis indicated the presence of leptin in bovine milk, while reverse transcription-polymerase chain reaction (RT-PCR) indicated the presence of leptin mRNA in mammary tissue and cultured bovine mammary epithelial cells (MAC-T cell line). A real time RT-PCR method was developed that allowed quantitative assessment of bovine leptin mRNA over approximately 3 orders of magnitude. Time course studies indicated a rapid increase in leptin mRNA in response to insulin or insulin-like growth factor-I (IGF-I). When normalized against bovine GAPDH as an internal control, 0.5 or 1h treatment with 10 ng/mL insulin gave 39+/-4 and 64+/-2-fold increase in leptin mRNA compared with 0 h control. Leptin mRNA was increased 257+/-9 and 75+/-23-fold by 0.5 or 1h treatment with 10 ng/mL IGF-I. Dose response studies indicated significant increases in leptin mRNA in response to as little as 1 ng/mL insulin or 0.1 ng/mL IGF-I. Maximum increase in leptin mRNA was observed in response to 10 ng/mL insulin and 10 ng/mL IGF-I. These results indicate that production of leptin by bovine mammary epithelial cells can be regulated by factors known to alter mammary function and nutrient partitioning. This suggests that leptin may be an autocrine/paracrine signal in the bovine mammary gland.