Vanadate inhibits protein chain initiation in rabbit reticulocyte lysates. The evidence that supports this conclusion is as follows: (i) the biphasic kinetics of inhibition in which protein synthesis is maintained at the control rate for 1-2 min is followed by an abrupt decline in the rate of synthesis; (ii) inhibition is associated with a marked disaggregation of polyribosomes and a concomitant increase in 80S ribosomes; and (iii) vanadate concentrations that inhibit protein chain initiation do not inhibit polypeptide chain elongation or the aminoacylation of tRNA. In partial reactions of protein chain initiation, vanadate concentrations that inhibit protein synthesis have no detectable effect on the formation of eukaryotic initiation factor eIF-2-promoted ternary complex with Met-tRNAf and GTP and on the assembly of 40S ribosomal subunit-Met-tRNAf complexes. On the addition of mRNA, the 40S ribosomal subunit-Met-tRNAf complexes also are transformed into 80S ribosome-mRNA-Met-tRNAf complexes, termed 80S initiation complexes. In vanadate-treated samples, however, these 80S initiation complexes are defective and unable to proceed beyond this step.