We present a colony assay system that allows in situ identification of human basophil/mast cell (basophil) colonies. In methylcellulose culture, in the presence of phytohemagglutinin-leukocyte conditioned media (PHA-LCM), human peripheral blood and bone marrow cells form colonies that can be distinguished by their unique morphological characteristics. Pure basophil colonies are diffuse, small colonies containing small, round, highly refractile cells. These characteristics of the constituent cells led us to the observation that a significant number of basophils are found in combination with eosinophils. The mixed eosinophil/basophil colonies have the distinctive elements of pure eosinophil and pure basophil colonies. Usually, these are diffuse colonies with compact clusters of slightly larger, darker-appearing cells. We also found colonies that contained basophils and neutrophils/monocytes, but this type could not be consistently identified by in situ morphology. Cytochemical analysis confirmed the metachromatic nature of the granules in the basophils. The presence of IgE receptors on the cells was documented by indirect immunofluorescent staining after passive sensitization with purified human IgE. Peripheral blood cells from six healthy volunteers formed 5.7 +/- 1.0 (mean +/- SEM) pure colonies in 2 X 10(5) cells. Cultures of bone marrow cells from patients with various types of anemia had 9.0 +/- 1.5 colonies in 10(5) cells. This is the first description of a colony assay system for in situ identification of a pure population of basophilic granulocytes.