A one-step non-radioactive assay to determine the proliferation of murine lymphocytes, lymphoid tumor cells and hybridoma cells is described. This assay requires the addition of Alamar Blue dye to cell cultures and the degree of change in its color, which is reflective of the extent of cellular proliferation, can be determined by an ELISA plate reader. Alamar Blue must be added during the initial phase of cell culture. The pattern of concanavalin A (ConA) or anti-CD3 antibody-induced proliferative response of murine lymphocytes as assessed by Alamar Blue was similar to that of a [3H]thymidine assay. Similarly, the spontaneous proliferation curve of anti-CD3 antibody secreting cell line (YCD3-1), monocytic macrophage cell lines (PU5-1.8, P388D1, J774.1) and myeloma cells (Sp2/0) as determined by Alamar Blue closely resembled that of the [3H]thymidine assay. The minimum detectable number of proliferating cells was comparable in Alamar Blue and [3H]thymidine assays. Since cell lysis/extraction and washing procedures are not involved in the Alamar Blue assay, this approach has several distinct advantages over currently available assays (eg. [3H]thymidine). First, it allows daily monitoring of proliferation without compromising the sterility of cultures. An indication of proliferation can be evaluated (spectrophotometrically or visually) as early as 24 h after ConA stimulation. Second, unlike previously reported assays, Alamar Blue permits further analysis of proliferating cells by other methods. Analysis of cells in culture with Alamar Blue for various surface antigens (CD44, CD45RB, CD4, heat stable antigen) by flow cytometry revealed that the fluorescent profile and relative percentage of cells in cultures with the Alamar Blue were comparable to those without this reagent. The salient advantages of Alamar Blue assay over the [3H]thymidine assay include: (i) non-radioactivity; (ii) simplicity; (iii) less costly; (iv) non-labor intensive; (v) rapidity of assessment of proliferation of large number of samples; (vi) non-toxicity; (vii) usefulness in determining the kinetics of cell growth of hybridomas; and (viii) non-interference of secretion of antibodies by a hybridoma cell line.