To investigate the roles of the amino acids of rat prolactin (rPRL), the structure of which is presumed to consist of an antiparallel, four-α-helix bundle, mutations constructed by site-directed mutagenesis were assayed in terms of their receptor binding and Nb2 cell proliferation activities. Replacement of P64L (replacing proline at position 64 with leucine) and K67E, which are located in the long loop region between helices 1 and 2, produced drastic decreases in the binding and proliferation activities. Mutations at D91 and E118 in the second and third helices, respectively, resulted in increased Nb2 cell proliferation activity with a slight increase in receptor binding activity. Mutations at L81 in the second helix and Y145 and W148 in the second loop between helices 3 and 4 produced no marked changes. Mutation at D158N in helix 4 markedly increased receptor binding activity with a slight loss of Nb2 cell proliferation activity, although two other mutations, D158H and D158R, produced a decrease of receptor binding activity without notable changes in Nb2 cell proliferation activity. These results demonstrate that P64 and K67 are crucial for PRL function while replacement of D91, E118 and D158 possibly leads to functional enhancement.