Traditional methods of diagnosing visceral leishmaniasis (kala-azar) in India suffer from a number of disadvantages. Amplification of multicopy nuclear genes and messenger ribonucleic acid of Leishmania by the polymerase chain reaction (PCR) was evaluated as an alternative assay under various clinical conditions. PCR of peripheral blood has the highest absolute sensitivity among all the available procedures, and is particularly useful for detecting parasites in early infections, post kala-azar dermal leishmaniasis, concurrent infections and immunocompromised cases, but is not so reliable for late infections. PCR of immunopurified blood mononuclear cells indicated the association of parasites with monocytes as well as non-monocyte cell types.