PG (prostaglandin) E1 inhibits the uptake of iridine, thymidine, 2-deoxy-D-glucose and L-isoleucine into human diploid WI38 fibroblasts. The inhibition occurs within seconds of the addition of the prostaglandin to the culture. PGE2, PGF1alpha and PGF2alpha behave similarly. Arachidonic acid and 8,11,14-eicosatrienoic acid also decrease uptake in the presence or absence of indomethacin. Other unsaturated fatty acids such as oleic acid, linoleic acid and linolenic acid are essentially inactive. Ricinoleic acid (the 9-hydroxyoleic acid), however, inhibits uptake to about the same degree, at concentrations similar to those of the prostaglandins. Results indicate that this rapid blockage by the prostaglandins and certain fatty acids is not cyclic AMP-mediated. For example, although PGF1alpha and PGF2alpha are much poorer stimulators of cyclic AMP formation than are PGE1 and PGE2, they are nevertheless effective inhibitors of substrate uptake. Adrenaline, a very effective stimulator of cyclic AMP formation in the cells, is not inhibitory. Also, the addition of 8-methylthioadenosine 3':5'-cyclic monophosphate (methylthio cyclic AMP) to the culture, methylthio cyclic AMP decreases the uptake of nucleotides into cultures undergoing active cell division, approximately to values found in quiescent cultures. PGE1 also has this effect on cells undergoing active growth. This gradual decrease is substrate uptake caused by PGE1 appears to be a separate event from its initial rapid inhibition of uptake.