Insulin-like growth factor-I (IGF-I), IGF-II, and the type 1 IGF receptor (IGF-IR) gene transcripts were localized in sections of ovine uterus collected from 53 ewes at different stages during the estrous cycle and the first 3 weeks of pregnancy using in situ hybridization with synthetic oligonucleotides. Binding studies on cryostat sections using [125I]IGF-I or [125I]des(1-3)IGF-I were carried out to assess whether binding sites for IGF-I colocalized with sites of IGF-IR gene transcription. Peak IGF-I mRNA concentrations occurred at estrus in both the periepithelial endometrial stromal cells and the longitudinal and circular muscle cells of the myometrium. Concentrations of transcript returned to basal levels by 48 h after estrus. The variations in IGF-I mRNA concentration during the estrous cycle showed significant correlations with the estradiol receptor concentration in the same cell types, measured in the same animals by immunocytochemistry (P < 0.01). IGF-II mRNA was localized exclusively to the caruncular stroma (high transcription) and endometrial stroma (low transcription), with levels showing no positive correlation to the concentration of estradiol receptors, progesterone receptors, or plasma progesterone, but decreasing gradually between estrus and days 14-15 (P < 0.05). IGF-IR mRNA was localized mainly to the deep and superficial glandular epithelium, with lower levels of transcription in the caruncular stroma and myometrium. Transcription in the deep glands remained high throughout the cycle, but in the superficial glands and myometrium, a small peak occurred in the early luteal phase (days 1-2), 24-48 h after peak IGF-I gene transcription. [125I]IGF-I- and des(1-3)IGF-I-binding sites colocalized with sites of IGF-IR gene transcription. Additional binding sites were revealed with the iodinated ligands in the myometrium, superficial glands, deep stroma, and blood vessel walls, which were probably attributable to the presence of binding proteins (IGFBPs). The different affinities of des(1-3)IGF-I for IGFBP-3, -2, and -1 lead us to suggest that IGFBP-2 and/or -1 are present in the deep stroma and glands, whereas IGFBP-3 may be responsible for the additional binding sites in the myometrium and blood vessel walls. Comparisons between pregnant and nonpregnant ewes on days 2-15 after estrus did not reveal any clear pregnancy-associated differences in either the sites or levels of IGF-I, IGF-II, or IGF-IR gene transcription.