The plasmid gene cat-86 specifies chloramphenicol-inducible chloramphenicol acetyltransferase in Bacillus subtilis. Induction by the antibiotic is primarily due to activation of the translation of cat-86-encoded mRNA. It has been suggested that the inducer stalls ribosomes at a discrete location in the leader region of cat-86 mRNA, which causes the destabilization of a downstream RNA secondary structure that normally sequesters the cat-86 ribosome binding site. It is the destabilization of this RNA secondary structure that permits translation of the cat-86 coding sequence. In the present report, we show that ribosomes that were stalled in the cat-86 leader by starvation of host cells for the amino acid specified by leader codon 6 induced gene expression to a level above that detected when cells were starved for the amino acids specified by leader codons 7 and 8. Starvation for amino acids specified by leader codons 3, 4, or 5 failed to activate cat-86 expression. These results indicate that the stalled ribosome that is most active in cat-86 induction has its aminoacyl site occupied by leader codon 6. To determine if chloramphenicol also stalled ribosomes in the cat-86 regulatory leader such that the aminoacyl site was occupied by codon 6, we separately changed leader codons 3, 4, 5, and 6 to the translation termination (ochre) codon TAA. Each of the mutated genes was tested for its ability to be induced by chloramphenicol. The results show that replacement of leader codons 3, 4, or 5 by the ochre codon blocked induction, whereas replacement of leader codon 6 by the ochre codon permitted induction. Collectively, these observations lead to the conclusion that cat-86 induction requires ribosome stalling in leader mRNA, and they identify leader codon 6 as the codon most likely to be occupied by the aminoacyl site of a stalled ribosome that is active in the induction.