Conditioned medium (CM) obtained from rat cerebellar astrocytes cultured in a serum-containing medium was able to inhibit [3H]thymidine incorporation into proliferating astrocytes, when compared to fresh medium. This effect could be attributed to two fractions of the CM with different molecular weights. The low molecular weight fraction (Mr less than 1,000) inhibited the cellular transport of the labeled precursor, without significantly affecting cell proliferation. The high molecular weight fraction (Mr greater than 10,000) showed a strong inhibitory effect on astrocyte proliferation, which was documented using different assay techniques: i) [3H]thymidine incorporation performed in conditions preventing the effects of CM on transport; ii) [3H]thymidine autoradiography; iii) determination of the DNA content of the cultures. The inhibitory activity was present in media conditioned by non proliferating astrocytes treated with the antimitotic cytosine arabinoside, but not in media conditioned by neuron-enriched cultures nor in a chemically defined (N2) CM. The antiproliferative activity of astrocyte CM could be due either to a rapid depletion of mitogenic factors present in serum, or, to a secretion of growth inhibitory factor(s) by cultured astrocytes.