Superoxide/hydrogen peroxide production by site IQin complex I of the electron transport chain is conventionally assayed during reverse electron transport (RET) from ubiquinol to NAD. However, S1QELs (specific suppressors of superoxide/hydrogen peroxide production by site IQ) have potent effects in cells andin vivoduring presumed forward electron transport (FET). Therefore, we tested whether site IQgenerates S1QEL-sensitive superoxide/hydrogen peroxide during FET (site IQf), or alternatively, whether RET and associated S1QEL-sensitive superoxide/hydrogen peroxide production (site IQr) occurs in cells under normal conditions. We introduce an assay to determine if electron flow through complex I is thermodynamically forward or reverse: on blocking electron flow through complex I, the endogenous matrix NAD pool will become more reduced if flow before the challenge was forward, but more oxidised if flow was reverse. Using this assay we show in the model system of isolated rat skeletal muscle mitochondria that superoxide/hydrogen peroxide production by site IQcan be equally great whether RET or FET is running. We show that sites IQr and IQf are equally sensitive to S1QELs, and to rotenone and piericidin A, inhibitors that block the Q-site of complex I. We exclude the possibility that some sub-fraction of the mitochondrial population running site IQr during FET is responsible for S1QEL-sensitive superoxide/hydrogen peroxide production by site IQ. Finally, we show that superoxide/hydrogen peroxide production by site IQin cells occurs during FET, and is S1QEL-sensitive.