We have investigated the relative amounts and sites of synthesis during the rat estrous cycle of the two ovarian mRNAs encoding the long and short PRL receptors (PRL-R). Quantitative analysis has revealed that the mRNA encoding the short PRL-R is consistently present throughout the cycle in lower quantities than the long receptor mRNA. Both receptor mRNAs are at maximal levels during proestrus, decline to their lowest level of expression during estrus, then gradually rise in metestrus and diestrus. By in situ hybridization, both receptor mRNAs are present during early proestrus in corpora lutea, in the granulosa cell layers of large Graafian follicles, and in the interstitial cells closely associated with these follicles. The short PRL-R mRNA was detected at significant levels in the granulosa-derived cumulus oophorus and in the thecal cell region at this time, whereas the long PRL-R mRNA was only weakly expressed in these cell types. In contrast, the long PRL-R mRNA was present at higher levels, compared to the short receptor mRNA, in the granulosa cells of preantral follicles in the interior of the ovary. On late proestrus, the long PRL-R mRNA was found predominantly in the mural granulosa cells of large Graafian follicles and in corpora lutea, but by estrous morning this mRNA appeared to be mostly restricted to the corpora lutea. This distribution was maintained through estrous evening and metestrous morning. On diestrus, both mRNAs were present in some corpora lutea and in the granulosa cell layer in a subset of the larger Graafian follicles, but were detected at even higher levels in the interstitial cells surrounding these follicles; again, the long receptor mRNA appeared to be only weakly expressed in the thecal cell region of these follicles. These results indicate that the levels and locations of PRL-R mRNA expression in the ovary, and therefore, the potential responsiveness of the ovary to PRL, change throughout the reproductive cycle. Furthermore, the presence of both receptor mRNAs in several different ovarian cell types suggests that both of these receptor forms play important roles in PRL physiology in the ovary.