In this study we report on the effect of glycerol used in storage buffers, on the inhibitory kinetic behavior of NADP(+) on rat kidney glutathione reductase (rkGR) stored at -80 degrees C in +/- 10% (v/v) glycerol. At fixed [GSSG] and varied [NADPH], rkGR stored +/- glycerol were inhibited competitively by NADP(+). Although the addition of glycerol decreased K-m (9.7 +/- 0.9 mu M) and Ki(NADp+) (26.6 +/- 2.2 mu M) for NADPH, the catalytic efficiency (K-cat/K-m = 2.75 x 10(7) M-1.s(-1)) was increased 1.64 fold. At fixed [NADPH] and varied [GSSG], rkGR exhibited noncompetitive (K-i = 397 69 mu M) and linear mixed-type (K-i = 59.6 +/- 8.4 mu M; K-s = 49.9 +/- 3.2 mu M; alpha = 7.74) inhibitions with and without glycerol during storage. The K-cat/K-m of rkGR stored in glycerol was 5.16 x 10(6) M-1.s(-1). Although the increase in K-cat was 1.25 fold, the K-cat/K-m was minimally affected (1.06).