Double immunoenzymatic method for sequential staining with two different monoclonal murine antibodies and two different enzymes was shown to be useful in defining hematopoietic cell subpopulations in human tissues and blood. The method allows for the identification, localization, and enumeration in the same section of distinct cell populations. Air-dried smears of cell mixtures can be stained. The optimal sequence of enzymes/substrates was: horseradish peroxidase/3-amino-9-ethylcarbazole followed by the alkaline phosphatase-anti-alkaline phosphatase complex/naphthol AS-MX phosphate. Red-and blue-colored reaction products are easy to view in a light microscope. Combinations of two different mouse monoclonal antibodies or of a mouse monoclonal antibody and polyclonal antiserum made in rabbits or goats can be sequentially applied to the same section or smear thus facilitating a definition of the distribution of two cell populations reactive with these antibodies. The relative distribution patterns in tissues of cells bearing distinctive antigens are important in studies of cellular differentiation and of human pathogenetic processes including neoplasia and transplant rejection.