Acute effect of prolactin (PRL) given intraperitoneally 1 h before on calcium fluxes was studied in 6 in situ intestinal segments from weaned, sexually mature and aged rats. In mature rats, PRL increased net passive calcium absorption in jejunum and cecum by 64% and 38%, respectively, by enhancing lumen-to-plasma calcium flux (CaL-P) in the jejunum and by reducing plasma-to-lumen flux (CaP-L) in the cecum. Since PRL-enhanced both CaL-P and CaP-L in the ileum, net calcium absorption did not change. In weaned rats, PRL significantly increased CaL-P in the jejunum but not in the ileum. However, the increase in net absorption was not significant. In contrast, effect of PRL was not seen in aged rats. By demonstrating an absence of PRL action on jejunal calcium fluxes when sodium-free test solution was used, we reconfirmed the sodium-dependent PRL action on passive calcium absorption. The PRL-induced parallel increase in the lumen-to-plasma transport of 45Ca and [3H]mannitol indicated that PRL action was to increase the paracellular transport of calcium.