Macrophages are the major cytokine producers in chronic inflammatory diseases, but the biochemical pathways regulating cytokine production are poorly understood. This is because genetic tools to dissect signaling pathways cannot be used in macrophages because of difficulties in transfection. We have developed an adenoviral technique to achieve high efficiency gene delivery into macrophages and recently showed that spontaneous TNF-α production in rheumatoid arthritis joint cells, chiefly from macrophages, is 75% blocked by adenoviral transfer of IκBα. In this report we use the same adenovirus to investigate whether the production of a number of proinflammatory cytokines (eg, TNF-α, IL-1β, IL-6, and IL-8) from human macrophages depends on NF-κB. While the cytokine response to certain inducers, such as LPS, PMA, and UV light, is blocked by overexpression of IκBα, the response to zymosan is not. In contrast, anti-inflammatory mediators (IL-10 and IL-1 receptor antagonist) induced by LPS are only marginally inhibited by IκBα excess. These studies demonstrate several new points about macrophage cytokine production. First, there is heterogeneity of mechanisms regulating both the proinflammatory and anti-inflammatory cytokines within populations of a single cell type. In addition, the results confirm the utility of the adenoviral technique for functional analysis of cytokine induction. The results also confirm that there are autocrine and paracrine interactions regulating cytokine synthesis within a single cell type. The selectivity of NF-κB blockade for proinflammatory but not anti-inflammatory mediators indicates that in macrophages, NF-κB may be a good target for the treatment of chronic inflammatory diseases.