Anterior pituitary cells were obtained from ovariectomized hamsters and cultured for 4 days. The cells were lysed and intracellular proteins, including FSH, were separated by polyacrylamide gel isoelectric focusing. FSH present within cell lysates so separated was quantitated by a specific FSH radioimmunoassay (RIA). Six distinct species of intracellular FSH were observed with pI values = 5.9, 5.7, 5.3, 5.1, 4.7 and 4.3 -- 3.8. All six species were detectable when proteins from a concentrate of incubation medium collected from those cell cultures were separated by isoelectric focusing. Similar isoelectric points were obtained when the FSH species present within an anterior pituitary homogenate obtained from ovariectomized hamsters were separated by the same technique. All but two of these six species of intracellular FSH were well recognized by a hamster FSH receptor preparation. However, these two species of FSH (pI values = 4.7 and 4.3 -- 3.8) do not appear to be beta subunits of the intact glycoprotein as determined by gel filtration. Thus, the present studies demonstrate that, as observed previously in anterior pituitary glands obtained from intact and castrated animals, pituitary cells that have been enzymatically dissociated and cultured in vitro for 4 days contain and secrete multiple species of FSH that are separable by polyacrylamide gel isoelectric focusing. This in vitro system may be useful for studying the hormonal factors that regulate FSH species synthesis and secretion.