Measuring the incorporation of radioactive thymidine into the cell nucleus gives important information as to cell activation and proliferation. In this study the DNA-intercalating fluorochromes, Hoechst 33342 and Hoechst 33258, were tested as an alternative to the classical [3H]thymidine assay. Mitogen and alloantigen stimulated lymphocytes as well as FK 506 and CsA inhibited lymphocytes were treated with the two dyes, and the cell number and proliferation rates by means of measured fluorescence values. Of these tested fluorochromes H33342 appears to be an appropriate alternative to the [3H]thymidine assay. It mirrors the cell number in a fast and convenient manner without any pretreatment of the cell suspension which can remain in the culture plates. The complete assay procedure including data analysis can be performed rapidly and the standard deviations are small. This dye may also prove to be of value in other assay procedures, e.g., adhesion experiments.