Independently of the stimulating agent used, generation of O2- by human neutrophils is transiently inhibited when the cells have been exposed to elevated temperatures. This phenomenon is concomitant with the synthesis of heat shock proteins (HSPs). We have investigated a possible relationship between HSPs and modulation of NADPH oxidase activity in human neutrophils exposed to heat. HSPs were not involved in the inhibition of O2- generation since 1) in enucleated cytoplasts, which are unable to synthesize proteins, the generation of O2- was inhibited after exposure to 43 degrees C, 2) using actinomycin D (Act D) in intact cells, it was possible selectively to inhibit the synthesis of HSPs without modifying the inhibition of NADPH oxidase activity that followed HS. Furthermore, the recovery of NADPH oxidase activity was not under the control of HSPs because the enzyme recovered as well in Act D-treated neutrophils. The NADPH oxidase activity was reconstituted in a cell-free assay by combining the cytosol with the plasma membrane-enriched fraction in the presence of arachidonic acid (AA) and NADPH. Subcellular fractions obtained from control or heated neutrophils exhibited similar oxidase activities suggesting that heat exposure did not induce denaturation of the oxidase components but rather altered the mechanisms of translocation and/or assembly of these components with the plasma membrane. This hypothesis was supported by the inhibition of the granule release in heated cells, a process which also requires translocation and association fusion with the plasma membrane. On the other hand, preexposure of neutrophils to HS prevented the inhibition of O2- generation during a second challenging HS. This acquired thermotolerance was abolished when the synthesis of HSPs was inhibited during the first HS with Act D, indicating a direct relationship between protection of O2- generation and synthesis of HSP. Here we demonstrate that synthesis of HSPs and inhibition or recovery of NADPH oxidase activity are concomitant but unrelated phenomena. In contrast, accumulation of HSPs in thermotolerant neutrophils appeared to play an important role in the prevention of NADPH oxidase inhibition. These results provide further insights into the behavior of human neutrophils and NADPH oxidase upon heat injury.