The tetramer core of 2'5'-adenylate was tested in vitro for its effect on erythroid colony-forming units (CFU-E) from bone marrow of adult rats. Inhibition of CFU-E was 2'5'-adenylate dose dependent, when tested with concentrations of 0.05- 1.0 mM. Also, 2'5'-adenylate was inhibitory when incubated with 2, 6 or 10 X 10(4) cells/0.1 ml plasma clot. At 0.1 mM concentration of 2'5'-adenylate there was a 30-50% reduction of erythroid colonies for each of these three cell concentrations. There was an early decrease in 3H-thymidine incorporation when 2'5'-adenylate was added to erythropoietin-stimulated bone marrow cell suspensions prior to plating. It is suggested that in situ production of 2'5'-adenylate may represent a regulatory mechanisms in red cell maturation.