Lindane (γ-hexachlorocyclohexane) is a commonly used pesticide that bioaccumulates in mammalian adipose tissue. Lindane inhibits gap junctional intercellular communication and oscillatory contractions of pregnant rat myometrium in vitro. The present study investigated the role of oxidative stress in lindane's inhibition of myometrial function in mid-gestation pregnant rat uteri. Lucifer yellow dye was microinjected into cultured myocytes to assess gap junctional intercellular communication. Lindane exposure (100 μM) resulted in a time-dependent, biphasic inhibition of dye transfer. This pattern of inhibition was also seen upon cell exposure to the pro-oxidant, tert-butyl hydroperoxide (100 μM). Lindane's initial and secondary-onset dye transfer inhibitions were reversed by cotreatment and pretreatment with the antioxidants, α-tocopherol (25–100 μM), diphenyl-1,4-phenylene diamine (10–30 μM), and superoxide dismutase (100–400 U/ml). d-mannitol (100–300 mM) also reversed lindane's initial dye transfer inhibition. Nitro blue tetrazolium reduction to formazan (measured spectrophotometrically) was elevated upon exposure of cultured cells to lindane or tert-butyl hydroperoxide, indicating the presence of reducing agents. Lipid peroxidation, assessed as thiobarbituric acid-reactive substances, was also elevated in lindane-exposed cell cultures. α-Tocopherol reversed this elevation. Finally, uterine contractility was assessed by measuring isometric contractions of uterine strips hung in standard muscle baths. Pretreatment with α-tocopherol prevented lindane's abolishment of uterine contractions in vitro. These data support the hypothesis that lindane inhibits uterine contractility and myometrial gap junctions by establishing an oxidative stress environment.