The present study investigated the importance of pulsatile vs. continuous protein kinase-C (PKC) stimulation in regulating pituitary gene expression. Adult female rat pituitaries were dissociated, and cells were plated for 48 h, then inserted into perifusion chambers (n = 5-8/group). Chambers received pulses of GnRH (100 pM) plus TRH (4 nM) or sn-1,2-dioctanoylglycerol (DOG; peak chamber concentration, 0.2, 1, or 5 mM; vehicle pulses to controls) every 60 min or a continuous infusion of phorbol 12-myristate 13-acetate (PMA; 20 nM). Secretory responses were determined in perifusate fractions collected after 2 and 22 h of perifusion. After 24 h of treatment, the cells were recovered, total RNA was extracted, and messenger RNAs (mRNAs) were measured by dot blot hybridization. The data revealed that GnRH plus TRH and both pulsatile (DOG) and continuous (PMA) PKC stimulation increased LH, FSH, TSH, and PRL secretory activity. Pulses of GnRH plus TRH increased PRL, alpha, TSH beta, and FSH beta mRNAs, but not LH beta mRNA. Pulsatile DOG only increased LH beta and PRL mRNAs, with maximal responses seen after the 1-mM dose for LH beta and the 0.2-mM dose for PRL. In contrast, PMA stimulated significant increases in alpha, LH beta, and TSH beta, but not PRL or FSH beta. These data show that alpha, TSH beta, LH beta, and PRL mRNA expression are regulated by PKC. Maximal increases are seen after continuous stimulation (via PMA), with the exception of PRL, which requires a pulsatile signal pattern. Thus, intermittent activation of PKC does not appear to play a major role in regulating pituitary gene expression.