The binding characteristics of iodinated growth hormones (human, 125I-hGH; bovine, 125I-bGH; rat, 125I-rGH) and iodinated ovine prolactin (125I-oPrl) to plasma membranes prepared from livers of adult male, adult female and pregnant female rabbits were determined. Competitive binding curves were compared and pH and cation dependency established. The study was designed to examine species differences in growth hormone binding to rabbit plasma membranes and to study the nature of hGH binding in comparison to a lactogenic (oPrl) and somatogenic (bGH) reference hormone. Human growth hormone and oPrl binding exhibited a pH optimum at pH 5, although significant binding occurred at pH 7. Bovine GH and rGH did not show a clear pH optimum, demonstrating significant binding from pH 5 to pH 7. Human GH and oPrl but not rGH and bGH binding was strongly influenced by cation concentration in incubation solution. Divalent cations (Ca++, Mg++) significantly increased specific hGH and oPrl binding at a concentration of 10 mM, whereas monovalent cations (Na+, K+) significantly increased binding at a concentration of 100 mM. Competitive binding curves with liver membranes from male and pregnant rabbits showed that the ED50 for hGH was less than for rGH and bGH binding to male and pregnant rabbit liver membranes and oPrl binding to male rabbit liver membranes. In every case, hGH was even more effective than the homologous unlabeled hormone. The relative effectiveness of the other hormones (oPrl, bGH and rGH) was consistent with expectations based on the lactogenic and somatogenic properties of the hormones. A 1000-fold excess of unlabeled hGH was found to be more effective in displacing 125I-hGH than a combined 1000-fold excess of both oPrl and bGH. A comparison of relative capacities of the three membrane preparations for 125I-hGH, 125I-oPrl, 125I-bGH and 125I-rGH showed that the capacities of the membranes were essentially the same for the somatogenic hormones (125I-bGH and 125I-rGH). The capacity of the membranes for 125I-hGH was 3-5 fold greater than for rGH or bGH. The relative capacity for 125I-oPrl was 3-5 fold less than for hGH in membranes from pregnant rabbit liver and negligible in membranes from female and male rabbits. The relative capacities of the membranes for 125I-hGH were 0.52, 0.74 and 0.76 percent/μg of membrane protein for membranes from pregnant female, female and male rabbits respectively. The combined relative capacities of the membranes for 125I-oPrl plus 125I-bGH were 0.35, 0.21 and 0.15 percent/μg of membrane protein for membranes from pregnant female, female and male rabbits respectively. The present study suggests that the binding of hGH to membranes from rabbit liver is not fully explained by its lactogenic and somatogenic properties. The existence of a receptor for hGH separate from that of bGH and rGH and oPrl is a distinct possibility.