Of the natural glycosaminoglycans tested, only heparin was a potent inhibitor of poly (U)-directed polyphenylalanine synthesis by rat liver ribosomes (50% inhibition at 10 mug/ml). A chemically oversulfated chondroitin sulfate was twice as effective, and another synthetic polyanion, sodium pentosan polysulfate was ten times more effective than heparin. Chondroitin-4,6-sulfate was inhibitory at very high concentrations (15 mg/ml) and heparan sulfate at concentrations above 100 mug/ml. The compounds interfere with the formation of ternary complex consisting of the ribosome, poly(U) and phenylalanyl-tRNA. The inhibitors prevented the attachment of the mRNA to the ribosome, probably by competition with poly(U) for the ribosomal binding site of mRNA. However, they were ineffective in doing so once phenylalanyl-tRNA has bound to the ribosomepoly(U) complex. Sucrose gradient analysis in presence of the inhibitors revealed a selective effect on the sedimentation of the small ribosomal subunit; the large subunit was unaltered. This effect, however, was dependent on the concentration of magnesium. In contrast to Escherichia coli ribosomes, no binding of the inhibitors to the particles could be demonstrated.