We have analyzed the 1 alpha, 25-dihydroxycholecalciferol [1,25(OH)2D3] receptor content of cultured cells from human amniotic fluid. Six cell lines were grown to confluence in a minimum essential medium containing 20% fetal calf serum. All had a normal karyotype, five were male and one was female. Hypertonic cytosol extracts were prepared by sonication followed by centrifugation at 200,000 X g 30 min. Saturation analysis was performed by incubating the extracts with [3H]-1,25(OH)2D3 (20-500 pM, 160 Ci/mmol) with and without 100-fold molar excess of unlabeled 1,25(OH)2D3. Linear sucrose gradient (5-20% w/v) analysis was performed with 1.5 nM [3H]-1,25(OH)2D3 alone or in presence of 100-fold molar excess, 1,25(OH)2D3. Functional responsiveness was measured by induction of 25-hydroxycholecalciferol-24R-hydroxylase with 1 and 10 nM 1,25(OH)2D3. The six cell lines studied had receptors with dissociation constant of 44 +/- 6 pM (mean +/- SEM). The binding capacity was 10,200 +/- 1,750 sites/ng protein (mean +/- SEM) with extreme values of 4,700 and 15,500. A single peak for specific binding migrating at approximately 3S was observed by sucrose gradient centrifugation. 25-Hydroxycholecalciferol-24R-hydroxylase was induced by 1 and 10 nM 1,25(OH)2D3 in a dose-dependent fashion. The data show that receptors for 1,25(OH)2D3 are present in cultured amniotic fibroblast-like cells early in pregnancy. These cells may thus prove to be useful for further characterization of 1,25(OH)2D3 receptors in fetal tissue.