The release of uncombined gonadotropin subunits by rat anterior pituitaries during an in vitro incubation as well as intracellular concentrations were assessed. Uncombined subunits and native gonadotropins were quantitated by radioimmunoassays after samples were subjected to gel filtration on Sephadex G-100 Superfine. Small, but detectable, amounts of uncombined rat LH beta subunit were released under basal conditions. GnRH increased the absolute amount of rLH beta released but did not alter the rLH beta/rLH molar ratio (approximately 0.02). Tissue extracts prepared in aqueous buffer (100,000 x g supernatants) and 0.5% Triton X-100 extracts of the 100,000 x g pellets from the initial homogenization ('pellet extracts') contained larger quantities of uncombined rLH beta as well as elevated rLH beta/rLH molar ratios (approximately 0.10 and approximately 0.20, respectively). Significant amounts of uncombined rLH alpha were released and were present in both tissue and pellet extracts. However, when FSH beta subunit was quantitated in tissue extracts after gel filtration, all of the immunoreactive materials eluted in the position of native rFSH (FSH beta/rFSH molar ratio less than 0.0025). Pellet extracts contained significant amounts of rLH beta, rLH and rLH alpha but lesser amounts of rFSH suggesting that intracellular gonadotropins are not completely extracted when homogenization is performed in aqueous buffers. Thus, rat anterior pituitaries contain and release significant amounts of the uncombined alpha subunit, relatively small amounts of uncombined rLH beta and extremely small amounts of uncombined FSH beta, if any.