Misidentification and cross-contamination of cell lines are major problems of cell cultures that can make scientific results and their reproducibility unreliable. This paper describes a PCR-based method for easily identifying or confirming the species of origin of cell lines by using a panel of oligonucleotides specific for the nine animal species most common in cell culture laboratories. A panel of 35 human and animal cell lines, whose species of origin were previously confirmed by isoenzyme assay, was studied with nine species-specific primer pairs that specifically anneal to DNA sequences codifying for human, cat, dog, mouse, rat, horse, rabbit, African Green monkey cytochrome c oxidase subunit I (cox I), and one primer pair specific for the cytochrome b gene of Chinese hamster. The amplified fragments were analyzed by electrophoresis in ethidium bromide-stained 2% agarose gels. The method is simple, rapid, highly sensitive, and useful for routinely monitoring the species identity of cell cultures.