When dispersed pituitary cells from 14-day-old female rats were sedimented in a bovine serum albumin gradient, a fraction was isolated which consisted of almost 90 percent of large cells that stained purple in the periodic acid-Schiff (PAS) reaction. Immunostaining indicated that over 85 percent of these PAS-purple cells were gonadotrophs containing both follicle stimulating and luteinizing hormone. Reproducible cell cultures could be obtained on poly-L-lysine coated dishes. As early as the second day in culture, the secretion of both follicle stimulating and luteinizing hormone was highly stimulated by luteinizing hormone-releasing hormone. The effect of FSH was as marked as that on LH. The data suggest that the isolated gonadotrophs are a specific functional subtype.