Using a polymerase chain reaction (PCR)-based strategy that distinguishes functional intron-containing genes from pseudogenes, the chromosomal location of the human intron-containing L35a ribosomal protein gene (rpL35a, HGMW-approved symbol RPL35A) was previously deduced to be onchromosome 18 using DNAs from a panel of human-rodent somatic cell hybrids. The inability to detect rpL35a in a human-hamster somatic cell hybrid containing only human chromosome 18 led to a reinvestigation of the chromosomal location of the human rpL35a gene. A clone containing intron 2 of rpL35a was isolated, sequenced, and used to isolate a P1 clone containing the human intron-containing rpL35a gene. By fluorescence in situ hybridization (FISH) analysis with the P1 clone as a probe, the rpL35a gene was located on chromosome band 3q29-qter. This location was confirmed by positive PCR analysis of a human-hamster somatic cell hybrid containing only human chromosome 3. The problem of using only human-rodent somatic cell hybrids for chromosome location is discussed.