The immunological and biological activities of growth hormone (GH) and prolactin (PRL) in the pituitary gland and serum of the squirrel monkey (Saimiri boliviensis boliviensis) have been studied. Proteins in pituitary homogenates were solubilized in 1% SDS, electrophoresed on 12% polyacrylamide gels, and transferred to nitrocellulose. Squirrel monkey GH and PRL were identified by immunoblotting with anti-human GH antibodies and a monoclonal antibody to ovine PRL, 6F11, respectively. Squirrel monkey GH appeared predominantly as two proteins of apparent molecular weight 22 and 20 kD, corresponding to native and variant forms of human GH. Squirrel monkey PRL appeared as two proteins of apparent molecular weight 24 and 26.5 kD, which co-migrated with native and glycosylated forms of ovine PRL. The cross-reactivity of neutralizing antibodies to human GH and PRL with squirrel monkey GH and PRL were examined using the Nb2 lymphoma bioassay. One of three monoclonal antibodies to human GH (2A1) neutralized squirrel monkey GH with an apparent affinity for squirrel monkey GH (IC50 = 70 ng IgG/ml) which was fourfold lower than for human GH (IC50 = 15 ng IgG/ml). Both polyclonal [AR38-5(1)] and monoclonal (9C3) antibodies to human PRL inhibited the activity of squirrel monkey PRL, athough their affinities for squirrel monkey PRL were four- and twentyfold lower than for human PRL. The activities of antibodies 2A1 and 9C3 on GH and PRL in squirrel monkey serum were also examined by the Nb2 bioassay. The anti-glucocorticoid RU486 was used in all incubations with squirrel monkey serum to eliminate the effect of high glucocorticoid levels on Nb2 cell growth. The mitogenic activity of squirrel monkey serum in the Nb2 assay was completely eliminated in the presence of 2A1 and 9C3. This study represents the first description of the biochemistry of GH and PRL in the squirrel monkey.