Immunoreactive rLH, rLH alpha, rLH beta and rFSH in the cell extracts and medium of rat anterior pituitary cell cultures were measured after gel filtration on Sephadex G-100 Superfine. Cell extracts contained uncombined rLH alpha and rLH beta present as approximately 40% and 4%, respectively, of native rLH on a molar basis. Unstimulated cultures appeared to release a large excess of rLH alpha (approximately 2560% of rLH on a molar basis) as well as a minimum of uncombined rLH beta. Stimulation of cultures with physiological (LHRH) or non-physiological agents (A23187, elevated K+) increased the absolute but not the relative quantities of uncombined subunits released relative to those present intracellularly. Thus, rat anterior pituitary cell cultures appear to produce and release an excess of free alpha subunit as well as a minimum of uncombined rLH beta. However, one cannot be certain whether the uncombined rLH beta represents a pool of 'free' rLH beta or simply results from the dissociation of rLH.