In rats with subcutaneously transplanted mammary carcinoma 5A (MC) and 2 to 10-fold elevations in the blood content of mature neutrophils, 30-50% of the neutrophils showed pronounced hypersegmentation. This phenomenon could be reproduced in liquid culture of bone marrow cells from normal (tumor-free) animals by 48 hr incubation with the MC host's serum, or with MC-conditioned medium whose activity was attributable to an over 50,000 MW protein. The effects in vitro, occurring without change in total cell number and accompanied by increases in gamma-glutamyltranspeptidase (GGT) and alkaline phosphatase (AP) activity, included decreases in the percents of progenitors (myeloblasts, promyelocytes, myelocytes, metamyelocytes and bands) and an increase in mature neutrophils 50% of which exhibited obvious hypersegmentation. Much less if any neutrophil hypersegmentation, and no statistically significant decrease in immature cells, occurred in response to the several colony stimulating factors (CSFs) tested, although (in addition to inducing GGT and AP) some CSFs did cause an increase in mature neutrophils. These investigations demonstrate the efficacy of a MC-elaborated blood-borne protein to promote myeloid cell maturation, and describe a system for the first time in which neutrophil hypersegmentation can be studied in vitro.