We constructed yeast strains containing deletion-insertion null alleles of the RPL16A or RPL16B genes encoding the 60S ribosomal subunit protein L16 to determine the role of L16 in the synthesis and function of ribosomes. Strains lacking a functional RPL16A gene grow as rapidly as wild type, whereas those containing a null allele of RPL16B grow more slowly than wild type. RNA analysis using RPL16 probes revealed that both RPL16 genes are transcribed and that RPL16B transcripts accumulate to twice the level of RPL16A transcripts. No evidence was obtained for the occurrence of dosage compensation at the level of RPL16 mRNA accumulation in either mutant. Strains lacking both RPL16 genes are apparently inviable, demonstrating that L16 is an essential yeast ribosomal protein. Introduction of an extra copy of either RPL16 gene into rpl16b mutants restored wild-type growth rates, indicating that the two forms of the L16 protein are interchangeable. rpl16 mutants are deficient in 60S ribosomal subunits relative to 40S subunits. 43S preinitiation complexes accumulate in half-mer polyribosomes in the absence of sufficient 60S subunits. We postulate that the slow-growth phenotype of rpl16 mutants results from the perturbation of initiation of protein synthesis.