We have previously shown that the mRNA for insulin-like growth factor-binding protein-4 (IGFBP-4) is present in adult rat ovaries, being localized predominantly to granulosa cells of atretic follicles. Now we have considered the following questions. What class of atretic follicles expresses IGFBP-4 mRNA? How does IGFBP-4 mRNA expression change during the estrous cycle? In keeping with our earlier work, a strong hybridization signal for IGFBP-4 mRNA was present in subpopulations of follicles throughout the estrous cycle. In all cases, the hybridization signal was localized to granulosa cells. Among the various types of follicles, IGFBP-4 mRNA was present almost exclusively in atretic graafian (antral) follicles. Morphologically, the outer layer of granulosa cells was positive, while cells in the cumulus oophorous were negative. By Northern analysis and in situ hybridization, the levels of IGFBP-4 mRNA were found to change over the estrous cycle. At 1000 h on proestrus (before the LH/FSH surge), the hybridization signal was relatively weak, being restricted in some (but not all) atretic Graafian follicles. At 2000 h on proestrus, (after the LH/FSH surge), essentially all atretic Graafian follicles were strongly positive for the message. The pattern of hybridization was similar at 0200 h on estrus, but the signal was less intense. At 1000 h on estrus, the hybridization signal was variable, ranging from very strong to weak or undetectable in atretic follicles. At this stage, however, the highest levels of IGFBP-4 mRNA were measured by Northern analysis; interestingly, a strong signal became apparent in the stromal cells. On diestrous day 1, the message levels decreased, and the signal was restricted to some atretic follicles. On diestrous day 2, the hybridization signal was very weak. There was virtually no detectable IGFBP-4 mRNA in any healthy follicle. In summary, we found that IGFBP-4 mRNA is 1) not detected in healthy dominant follicles; 2) localized almost exclusively to atretic Graafian follicles, except on estrus when it also appears in stromal cells; 3) localized predominantly to the mural granulosa cells in atretic follicles; and 4) undergoes changes during the cycle, being most prominent around estrous morning. The possibility that IGFBP-4 plays a role in the cyclic destruction of cohort Graafian follicles at estrus, perhaps by mechanisms involving hormones, is discussed.