We have recently shown that the posterior pituitary (neurointermediate lobe) contains a potent prolactin (PRL)-releasing factor (PRF) which is distinct from known PRL secretagogues. Posterior pituitary PRF appears to be a small peptide of an unknown cellular origin. Using pituitary stalk-sectioned (SS) male rats, the objectives of this study were: (1) to determine if PRF is transported from the hypothalamus or is synthesized within the pituitary gland, and (2) to compare changes in PRF activity with alterations in the posterior pituitary content of β-endorphin (β-END), oxytocin (OXY), and dopamine (DA). One or two weeks following pituitary SS or sham surgery (SHAM), acid extracts of the posterior pituitary and medial basal hypothalamus (MBH) were analyzed for their hormone content. PRF activity was assessed by determining the stimulation of PRL secretion from perifused anterior pituitary cells, DA was measured by HPLC, and OXY and β-END levels were determined by RIAs. OXY and DA concentrations in the posterior pituitary were reduced more than 95% at both 1 and 2 weeks after SS. PRF activity in the posterior pituitary was significantly reduced by 75 and 90%, 1 and 2 weeks after SS, respectively. In contrast, β-END levels in the posterior pituitary at these times increased 20 and 60%, as compared to SHAM rats. Unlike the posterior pituitary, OXY levels in the MBH increased 123% 1 week following SS, and 1,260% at 2 weeks. These increases may reflect the accumulation of OXY-containing secretory vesicles in the severed nerves. DA concentrations in the MBH showed a biphasic pattern. DA levels were initially decreased by 70%, and then increased, but remained 30% below SHAM levels. The reason for these alterations in DA levels is not clear. It is concluded that: (1) similar to OXY and DA, but unlike β-END, PRF is likely transported from the hypothalamus to the posterior pituitary, and (2) the low levels of PRF activity in the MBH suggest that PRF may be synthesized as a biologically inactive precursor, the amount of PRF in the MBH is too low to detect, or inhibitors of PRL secretion mask the PRF acti