Pediatric and adult tuberculosis (Tb) have been on the rise in recent years. In New York City there has been a 300% increase in the incidence of Tb among children 15 years and younger from 1987 to 1991. 1.3 million cases of pediatric tuberculosis occur yearly in developing countries. The diagnosis of Tb presents a particular problem in children due to the low yield of isolates of M. tuberculosis (MTb) from clinical specimens. We developed an ELISA spot assay using anti-MTb monoclonal antibodies (mAbs). The assay is designed to improve the diagnosis of Tb in children and adults. We generated 3 monoclonal antibodies against the surface of MTb (2 IgM and 1 IgG3) using routine hybridoma techniques. All 3 Mabs bind the surface of MTb as demonstrated by whole cell ELISA, indirect immunofluorescence and immunoelectronmicroscopy. They recognize different surface epitopes (LAM, mAPG and a protein moiety) and they show differences in cross reactivity patterns when incubated with different mycobacterial strains. The assay we designed uses 2 mAbs; it was designed as a capture ELISA, using a broadly cross reactive IgM mAb to capture the organisms from serum and a more selective IgG3 as the detecting mAb. By using a non-soluble color developing reagent and a specially designed chamber slide, single organisms can potentially be captured from fluids and directly visualized using light microscopy. The assay is simple to perform, can be done using existing equipment of any microbiology laboratory and does not require live organisms.