Abstract A principal chemical requirement by sparsomycin to inhibit protein synthesis was investigated in animal and bacterial (poly(U)-directed) cell-free systems. Peptide-bond formation was studied by the puromycin reaction and by chromatographic analysis of the polymerization products. It was found that sparsomycin does not effectively inhibit the puromycin reaction when phenylalanyl-tRNA is used as a substrate, whereas a definitely stronger inhibition occurs with acetylphenylalanyl-tRNA as a substrate. The analysis of the ribosome-bound polymerization products showed that sparsomycin induced an accumulation of diphenylalanine with the first substrate but not with the second. It is concluded that sparsomycin, to inhibit peptide-bond formation, necessarily requires that the tRNA in the donor ribosomal site does not carry an aminoacyl with a free amino group.