Different data have suggested that the density-dependent inhibition of growth in normal 3T3 cells is due to the release of inhibitory factor(s) into the medium. The present results provide more evidence supporting this hypothesis. From conditioned medium of dense cultures of 3T3 cells, we fractionated a thermolabile inhibitory diffusible factor (I.D.F.) with a molecular weight of about 40.000 This factor decreased the [11C]-inosine incorporation into purine nucleotides and into nucleic acids of stimulated 3T3 cells. DNA synthesis, determined by [11C]-thymidine incorporation into nucleic acids, and protein synthesis (determined by the protein content of the cultures maintained in the presence or absence of I.D.F.), were also decreased. The possibility that I.D.F. is involved in density-dependent inhibition of growth is discussed.