We describe a method to obtain results for immune status monitoring that uses a three-test panel, comprised of isotype control and 2 specific Mab tests (CD4/CD8/CD3 and CD16/CD19/CD3), in conjunction with a flow cytometer that directly measures absolute counts. Automated software is used for lineagespecific gating of three-color immunofluorescence to determine lymphocyte and lymphocyte subset counts. The autogating function of this software is shown to yield equivalent results to manual analysis by an expert user, and to be effective when as few as 25 target cells are present. The software is also shown to perform automatic quality control checks of the sample preparation, reagent, and automated analysis. We demonstrate that the sum of T (CD3 +), B (CD19 +), and natural killer (NK, CD16 + CD3−) cells, as a determination of all lymphocytes, correlates well with lymphocytes measured using a light scatter differential. Moreover, T + B + NK lymphocyte count is shown to be less error-prone than lymphocyte count from light scatter differential, and to minimize errors that arise from between-technician variation in sample preparation. Our data suggest that the new approach that we describe could offer an alternative to the traditional two-stage methods for measuring absolute counts of lymphocyte subsets for immune status monitoring. As such this method could reduce, through objective automated analysis, testing cost and complexity, without sacrificing the quality of results. © 1995 Wiley-Liss, Inc.