In this study, we have tested the hypothesis that unmodified prolactin (U-PRL) and phosphorylated prolactin (P-PRL) have differential roles in the autoregulation of PRL secretion in vivo. Recombinant human U-PRL and a molecular mimic of P-PRL (S179D PRL) were administered to male rats and to female rats in different physiological states and the effect on rat PRL release was measured. Administration of U-PRL elevated rat PRL in all female animals, but was without effect in males. By contrast, S179D PRL was inactive in females, but inhibited PRL release in males. Morphometric and immunohistochemical analyses demonstrated acidophil hypertrophy and evidence of increased PRL secretion in the pituitaries of U-PRL-treated females. Analysis of the two forms of PRL during prolactinoma induction in two differentially susceptible strains of rats found a strong temporal correlation among increased ratios of U-PRL: P-PRL, increased circulating PRL, and increased cell proliferation. We conclude (1). that the autoregulatory mechanism(s) can distinguish between the two major forms of PRL and that higher proportions of U-PRL not only allow for higher circulating levels of PRL, but are also autostimulatory, (2). that the autoregulatory mechanism( s) are set differently in males and females such that females are more sensitive to autostimulation by U-PRL and less sensitive to inhibition by P-PRL, and (3). that U-PRL and P-PRL may also have differential roles in the regulation of pituitary cell proliferation.