Measures of immune function have become increasingly important as endpoints in AIDS clinical trials, with respect to both modulation and reconstitution of immunity by experimental therapies. Measurement of immune function in this setting requires the development of robust analytic approaches suitable for the clinical laboratory. Experiments were performed to evaluate the suitability of using cultured heparinized ("whole") blood for induction of tumor necrosis factor alpha (TNF-) and gamma interferon (IFN-), two cytokines critical in AIDS pathogenesis. TNF- expression ranged from 229 to 769 pg/ml in lipopolysaccharide (LPS)-stimulated cultures and was not detected in unstimulated cultures. IFN- expression ranged from 0 to 112,000 pg/ml in phytohemagglutinin A (PHA)-stimulated cultures and from 0 to 789 pg/ml in antigen-stimulated cultures. The mean coefficient of variation observed in three weekly determinations was 0.47 for TNF- and ranged from 0.12 to 1.73 for IFN-. These values indicate that sample sizes of 8, 24, and 29 subjects would be sufficient to detect twofold changes in LPS-induced TNF- and in PHA- and antigen-induced IFN-, respectively, if two baseline and two treatment determinations were obtained, and if the interpatient variability of changes in true levels from baseline to follow-up is negligible compared to the variability in the three weekly measurements. Measurement of LPS-induced TNF- and mitogen- or antigen-induced IFN- can be performed simply and reproducibly in human immunodeficiency virus-infected persons by the whole-blood culture method. Further studies are warranted to determine the effect of overnight shipping on assay reproducibility and to determine the extent to which responses can be reliably detected in subjects with low CD4 cell numbers.