Pseudomonas aeruginosa is a gram-negative bacterium that causes both acute and chronic lung disease in susceptible patient populations. P. aeruginosa secretes numerous proteins and secondary metabolites, many of which have biological effects that likely contribute to disease pathogenesis. An unidentified small molecular weight factor was previously reported to increase IL-8 release both in vitro and in vivo. To identify this factor, the < 3 kDa fraction from P. aeruginosa-conditioned medium was subjected to HPLC analysis. A peak fraction that stimulated IL-8 release was found by mass spectrometry to have a MW of 224 Da. Based on this MW and other biochemical properties, we hypothesized that the factor was phenazine-1-carboxylic acid (PCA). Subsequent studies and comparison with purified PCA confirmed this hypothesis. Purified PCA exhibited a number of biological effects in human airway epithelial cells including increasing IL-8 release and ICAM-1 expression, as well as decreasing RANTES and MCP-1 release. PCA also increased intracellular oxidant formation as measured by electron paramagnetic resonance (EPR) and by an intracellular oxidant-sensitive probe. Antioxidants inhibited PCA-dependent increases in IL-8 and ICAM-1 suggesting that oxidants contributed to these effects. However, in contrast to the related phenazine compound, pyocyanin, PCA did not oxidize NAD(P)H at physiologically relevant pH providing preliminary evidence that PCA and pyocyanin may have distinct redox chemistries within the cell. Thus, PCA is a biologically active factor secreted by P. aeruginosa that has several activities that could alter the host immune and inflammatory response and thereby contribute to bacterial disease pathogenesis.