In addition to the pituitary, prolactin (PRL) in humans is produced at non-pituitary sites where it acts as a cytokine. We previously reported that PRL is expressed and released from breast adipose explants, raising the question as to the dynamics of its production and its regulation. Preadipocytes were isolated from breast adipose tissue obtained during breast reduction. PRL expression was transiently increased during early preadipocyte differentiation. Both isoproterenol, a beta-adrenergic receptor agonist, and PACAP, pituitary adenylate cyclase activating peptide, increased PRL expression, and release from preadipocytes. This stimulation was suppressed by several protein kinase inhibitors, suggesting involvement of multiple signaling pathways. Transfection of preadipocytes with a superdistal PRL promoter/luciferase reporter revealed two stimulatory domains and an inhibitory domain. These data establish the transcriptional regulation of adipocyte PRL by the superdistal PRL promoter, its transient expression during adipogenesis, and the stimulatory effect of catecholamines and PACAP.