Human PMN (non-adherent: density greater than 1.070 g/cm3) were used to prepare cell-free colony inhibiting activity (CIA). Intact PMN, PMN conditioned media (serum-free or serum-containing) and supernatant from immediately lysed PMN equally reduced the numbers of spontaneous bone marrow (density less than 1.070 g/cm3; depleted of PMN) colonies and clusters. Addition of CIA reproducibly reduced colony and/or cluster numbers by 35-70%. CIA was heat labile being inactivated at 37 degrees C. Daily additions of CIA reduced colony numbers even further; inhibition was dependent on [PMN] used to prepare CIA and on the potency of stimulation produced by colony stimulating cells (CSC). PMN from some patients with leukemia were inactive as inhibitors and did not condition media with CIA. All experiments were thereafter controlled by parallel assessment of supernatant of lysed PMN not demonstrating any CIA. CIA appears to be not only stored in PMN but released from viable, as well as degranulating PMN. Although not species-specific, CIA was specific in its nontoxic action on CSC; colony forming cells and cell-free colony stimulating activity were not affected. The influence of CIA on CSC could be reversed within 10 minutes, but not within 35 minutes of incubation. CIA may have possible physiological relevance.