Plasma from normal human subjects was subjected to a 3-phase methanol extraction. An increase in prolactin release from rat anterior pituitary tissue in vitro obtained with increasing doses of the plasma extract, while no increase in TSH release occurred. Control studies with an extract of simulated plasma using incubation medium with human serum albumin resulted in less release than a comparable plasma extract dose. The plasma extract did not lose activity with heating to 100 degrees C for 10 min, and incubation of normal plasma at 37 degrees C for 4 h prior to methanol extraction resulted in no loss of prolactin releasing activity. The plasma extract was subjected to cascade ultra-filtration through UM-10, UM-2, and UM-0.5 filters with no significant loss of prolactin releasing activity. Finally, the extract contained no TRH immunoreactivity. These results support the existence of a heat stable, ultra-filterable circulating PRF in human plasma. The origin of this material is unknown.