In order to gain insight into the conservation of determinants for tRNA identity between organisms, Schizosaccharomyces pombe and human amber suppressor serine tRNA genes have been examined for functional expression in Escherichia coli. The primary transcripts, which originated from E. coli plasmid promoters, were processed into mature tRNAs, but they were poorly aminoacylated in E. coli and thus were nonfunctional as suppressors in vivo. However, coexpression of cloned Saccharomyces cerevisiae seryl-tRNA synthetase led to efficient suppression in E. coli. This shows that some, but not all, determinants specifying the tRNASer identity are conserved in evolution.