Progesterone production and release in vitro, and mRNA expression for key steroidogenic enzymes, were studied in luteal tissue recovered in the immediate postovulatory period from cyclic gilts allocated to one of three treatments: moderate feed restriction during the first (RH) or second week of the estrous cycle, with (HR I) or without (HR) concomitant injections of long-acting insulin. Time of feed restriction affected neither progesterone production or release, nor mRNA expression for several key steroidogenic enzymes. However, luteal tissue from RH but not from HR gilts responded to LH stimulation by increasing progesterone production and release (P < 0.05). Insulin treatment increased progesterone production and release, restored luteal tissue responsiveness to LH, up-regulated steroidogenic enzyme mRNA expression, and down-regulated the tissue inhibitor of metalloproteinase-I mRNA expression in HR I compared with HR gilts (P < 0.05). In vitro progesterone production and gene expression were affected by time of tissue collection after ovulation in RH and HR gilts but not in HR I gilts, and were correlated with temporal changes in oviductal and peripheral plasma progesterone concentrations. Inherent differences in luteal function therefore appear to mediate latent effects of nutrition and insulin treatment on circulating progesterone concentrations in the critical postovulatory period in gilts.