The detection with lucigenin under physiological conditions is selective for ${cdot} O_{2}^{-}$, for it can be accepted that lucigenin indicates actual intramembranal $cdot O_{2}^{-}- formation$. Lucigenin chemiluminescence (CL) was elicited from the plasma membrane (PM) only by addition of reduced pyridine nucleotide. NADPH was preferred to NADH in PM and hepatocytes. This specificity was masked by $NAD(P)^+$ inhibition. The half maximum rate of CL increase was obtained with 1.5 ${mu}m$ NADH or 55 ${mu}m$ NADPH in hepatocytes and 6 ${mu}m$ NADH or 30 ${mu}m$ NADPH in plasma membranes. Measurement of these NADPH values required the presence of a NADPH-regenerating system. With NADPH the maximal rate obtained was 10 fold higher than with NADH. NADPH and NADH could produce CL when having access from either side of the membrane. They seemed to react with the identical acceptor because NADH-induced CL was also inhibited by $NADP^+$. The characteristics of ${cdot}O_{2}^{-}-formation$ produced by pyridine nucleotide will be discussed.