Rat anterior pituitary cells were dissociated with Pronase and trypsin. Recovery of Pronase—dissociated cells ranged from 33 to 38% based on LH and protein assays. Cell viability was 97% as determined by uptake of trypan blue and eosin Y. The dissociated cells were separated into 7 fractions of different volumes by velocity sedimentation at unit gravity for 4 hr. Radioimmunoassay for LH, bioassay for FSH, and electron and light microscopy were used to assess the degree of cell separation. Gonadotropic cells were found primarily in combined fractions (CFr) 1 and 2. These 2 fractions, which had 7% of the total cells recovered, had over 58% of the total LH activity. CFr 4 consisted largely of GH cells while CFr 6 had mostly chromophobic cells. The dissociated cells showed an increased uptake of 3H glucosamine with time, and CFr 1 and 2 showed the highest uptake. These 2 fractions also produced a substantial amount of LH which was found in the culture medium during a 3–day incubation Endocrinologyperiod. ( 92: 1639, 1973)