Burkholderia pseudomallei is a free-living organism that causes the potentially lethal tropical infection melioidosis. The disease is endemic in many parts of eastern Asia and northern Australia. The presence of two distinct biotypes in soil can be reliably distinguished by their ability to assimilate L-arabinose. Whereas some soil isolates could utilize this substrate (Ara+), the remaining soil isolates and all clinical isolates tested so far could not (Ara-). Only the Ara- isolates were virulent in animal models. We have raised a murine monoclonal antibody (MAb) that can readily distinguish Ara- from Ara+ biotypes. The MAb reacted with a high molecular weight component present only on the Ara- biotype. With this MAb, clinical and soil Ara- isolates gave identical positive reactions in agglutination, immunofluorescence, ELISA and immunoblot assays. Using these same assay systems, the soil Ara+ biotype did not react with the MAb. Similar but distinct immunoblot patterns were also noted when these two Ara biotypes were probed with sera from patients with melioidosis or with polyclonal immune rabbit sera. These data showed that the Ara- biotype from both clinical and environmental isolates is antigenically different from its Ara+ environmental counterpart. The SDS-PAGE protein and lectin-binding profiles of both groups of Ara- isolates were also found to be different from those of the Ara+ biotype.