The biologic and clinical heterogeneity of the various subtypes of non-Hodgkin's lymphoma is related to differences in morphologic, immunologic, and kinetic properties. Comprehensive studies characterizing these features in lymphomatous effusion have yet to be reported. We recently studied 27 effusion specimens from 26 patients with clinically suspected or confirmed lymphoma. Wright-Giemsa- and Papanicolaou-stained cytologic preparations, acridine orange nucleic acid flow cytometry, and immunoperoxidase staining of cell suspensions using antibodies to a battery of T and B cell markers were evaluated and compared with prior histologic accessions. Specimens were classified by cytologic characteristics according to the International Working Formulation Scheme and by acridine orange nucleic acid flow cytometry using the parameters of DNA, RNA, and proliferative activity. Correlation of the cytometric and morphologic data demonstrated that with increasing cytologic grade of lymphoma, the proliferative activity increased progressively and distinguished between grades (P less than 0.01). Immunologic studies identified B cell phenotype in 16 specimens, T cell in three, and true histiocytic lymphoma in one; one lymphoma had no cell markers (null cell). Six effusions proved to be inflammatory and reactive according to surface marker studies. Classification by cytologic characteristics showed good correlation with histologic classification performed previously. Immunologic study of cytologic specimens gave results identical to those achieved by frozen-section immunohistologic examination. Thus, immunologic and cytometric parameters can be readily performed on effusion specimens and aid in the diagnosis and classification of lymphomas.