A short term in vitro test for granulocyte chalone activity eas examined for its specificity and reliability. The test used the inhibition, by granulocyte extracts, of 3H-thymidine (3H-Tdr) uptake in to the acid-insoluble material by rat bone marrow cells in vitro to measure possible chalone activity. Among the many possible 3H-Tdr artifacts pool size dilution by Tdr contained in the extracts was excluded using an E. coli mutant requiring thymine. Several amino acids and biogenic amines do not affect the test. However, continuous and pulse labelling of bone marrow cells with 3H-Tdr, viability tests and micro flow fluorometric measurements of the cell cycle distribution following colcemid treatment strongly suggests that the cells do not proliferate in vitro during short term incubation, since practically no cells enter the S-phase, cells in the S-phase die and few if any cells proceed through G2 and mitosis. Moreover, the test cannot exclude cytotoxicity. Thus, the in vitro test may only sceem for an unspecific S-phase inhibitor and must hence be supplemented by another assay to prove the chalone nature of an extract or fraction. The test per se fails to meet most of the requirements of a valid granulocyte chalone assay.