The elucidation of heat tolerance mechanisms is required to combat the challenges of global warming. This study aimed to determine the antioxidant enzyme responses to heat stress, at the enzymatic activity and gene expression levels, and to investigate the antioxidative alterations associated with heat tolerance in the stems and roots of squashes using three genotypes differing in heat tolerance. Plants of heat-tolerant C. moschata, thermolabile C. maxima and moderately heat-tolerant interspecific inbred line Maxchata genotypes were exposed to moderate (37 degrees C) and severe (42 degrees C) heat shocks. C. moschata exhibited comparatively little oxidative damage, with the lowest hydrogen peroxide (H2O2), superoxide (O-2(-)) and malondialdehyde (MDA) contents in the roots compared to stems, followed by Maxchata. The enzyme activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD) were found to be increased with heat stress in tolerant genotypes. The significant inductions of FeSOD, MnSOD, APX2, CAT1 and CAT3 isoforms in tolerant genotypes suggested their participation in heat tolerance. The differential isoform patterns of SOD, APX and CAT between stems and roots also indicated their tissue specificity. Furthermore, despite the sequence similarity of the studied antioxidant genes among C. maxima and Maxchata, most of these genes were highly induced under heat stress in Maxchata, which contributed to its heat tolerance. This phenomenon also indicated the involvement of other unknown genetic and/or epigenetic factors in controlling the expression of these antioxidant genes in squashes, which demands further exploration.