The detection of antigen in the urine is increasingly being used for diagnosis of parasitic infections. A urinary antigen has recently been demonstrated in visceral leishmaniasis (VL), using a latex agglutination test. The results of our study show that the detected antigen is: heat-stable, precipitates with acetone and ethanol but not TCA, is sensitive to periodate and acid hydrolysis but not to pronase E, lipase, or neuraminidase. The antigen is a low molecular weight glycoconjugate that can be extracted by phenol-water, partitions into the aqueous phase when extracted with Triton X-114 or chloroform/methanol, and can be labelled by biotin hydrazide. Since this urinary antigen cannot be characterised by conventional SDS-PAGE and Western blotting, we used an affinity transfer blotting system in which antigens were captured onto nitro-cellulose paper previously coated with a specific antibody. Using this system a low molecular weight antigen (LMWA) spanning an area of the nitro-cellulose membrane corresponding to molecular weight of 5-20 kDa was detected in the urine of VL patients (from Nepal, Sudan, Brazil, Yemen and Spain) and of experimentally infected animals. No LMWA was detected in the urine of patients with malaria, schistosomiasis, or other nonparasitic diseases including typhoid and brucellosis. Immunoprecipitation, using antibody-coated latex, followed by immunoblotting showed that the LMWA is the target antigen in the previously described latex agglutination test ('KATEX'). The antigen is detectable in both the promastigote and amastigote stages of the parasite. Monoclonal antibodies (mAbs) against Leishmania glycoconjugates strongly react with this molecule. These results suggest that the detected antigen is highly specific and diagnostic for VL.