A Lactobacillus bulgaricus gene encoding a serine tRNA with the anticodon CGA was isolated from a L. bulgaricus clone bank and characterized. This gene is expressed and active in Escherichia coli. The wild-type form of the gene allele specifically complements the E. coli leuB6 mutation. This process depends on gene copy number; high copy number restores leucine prototrophy, while low copy number does not. We suggest that restoration of activity of the mutant leuB6 allele occurs by missense suppression. The L. bulgaricus tRNA(CGASer) when overproduced in E. coli is misacylated at a low frequency, leading to the insertion of an amino acid other than serine in response to the presumed mutant codon UCG in the leuB6 gene. Nucleotide (nt) sequences flanking the tRNA coding region are present in the L. bulgaricus tRNA gene, closely resembling E. coli promoter and terminator elements. A noteworthy feature of this tRNA gene is the extreme length (22 nt) of its extra arm. The 3'-terminal CCA of the tRNA is not encoded in this tRNA gene and thus must be added posttranscriptionally.