alpha-, gamma-, and delta-Hexachlorocyclohexane (HCCH), but not the beta-isomer, are shown to be potent stimuli for the production of superoxide anion (O2-) and the release of calcium in human polymorphonuclear leukocytes (PMNs). Although O2- production occurs in the absence of exogenous divalent cations, calcium (0.5 mmol/L) enhances O2- production by 50%. In addition, gamma-HCCH-induced O2- production is sensitive (IC50–30–40 mumol/L) to inhibition by the putative intracellular antagonist, 8-(diethylamino)octyl-3,4,5- trimethoxybenzoate hydrochloride (TMB-8). Furthermore, it is shown that gamma-HCCH induces a marked loss of membrane-associated calcium as monitored by chlortetracycline fluorescence. This pool of calcium appears to encompass the same pool released by the formylated tripeptide, formylmethionylleucylphenylalanine (FMLP). We suggest that the HCCHs represent a new class of stimuli of O2- production and calcium mobilization in PMNs.