We have tested the reliability of a standard IL-2 microassay (3H-thymidine uptake by IL-2-dependent T cell lines) as a measure of the colony promoting activity (CPA) required for PHA-induced T cell colony growth in semi-solid cultures. Colonies were obtained from freshly isolated mononuclear cells (PBL) (primary colonies) or pooled cells of primary colony (secondary colonies). PHA-stimulated PBL generated primary colonies in the absence of exogenous CPA which, however, when added to the cultures enhanced colony growth. In contrast, primary colonies failed to form PHA-induced secondary colonies in the absence of exogenously supplied CPA. There was a close correlation between the colony forming capacity of PBL and primary colonies from the same donors plated in the presence of added CPA (r = 0.98). When CPA was measured in the secondary colony growth assay and the data compared to IL-2 measurement in the standard IL-2 microassay, CPA and IL-2 levels did not correlate, suggesting that IL-2 and CPA may represent distinct factors. The usefulness of primary colonies as target cells for the measurement of CPA levels in a variety of conditioned media is discussed.