Nocardia lysozyme digest (NLD) and Nocardia water-soluble mitogen (NWSM) are two fractions derived from Nocardia opaca. In this report, we demonstrated that both fractions elicited significant secretion of tumor necrosis factor-alpha (TNF-alpha) in human monocytes. Supernatants from monocytes stimulated with NWSM and low concentrations of NLD displayed a cytotoxic activity against TNF-alpha-sensitive L929 cells, but supernatants from monocytes stimulated with high concentrations of NLD failed to lyse L929 cells. This latter phenomenon might be related to the secretion of an inactive form of TNF-alpha or the release of an inhibitor of TNF-alpha cytotoxic activity. Since it is well established that protein kinase C (PKC) plays a major role in the signaling of several monocyte activators, we investigated the putative role of PKC in cytokine synthesis induced by NLD and NWSM fractions. TNF-alpha secretion in response to both Nocardia fractions was inhibited by sphingosine, staurosporine and calphostin C, known PKC inhibitors, as well as by a PKC depletion procedure. In addition, NLD and NWSM induced a transient increase in [3H]phorbol dibutyrate binding, which assessed the activation of PKC. The data suggest the involvement of PKC in the signaling of NLD and NWSM fractions leading to the synthesis and the secretion of TNF-alpha by human monocytes.