A pot culture experiment was conducted with black gram [Vigna mungo (L.)Hepper] cultivars during summer season of 1999 under natural day light conditions to study the influence of some critical factors viz., Infiltration media components of Nitrate Reductase Activity (NRA) assay, diurnal fluctuations of NRA, distribution of NR in different plant parts and the physiological age of leaf, for determining the optimal NR activity. The results revealed that the standard infiltration media contain 1% v/v n-propanol, 0.05% v/v triton-X-100 and 100 mM potassium nitrate. NR activity significantly varied with photoperiod. Among different plant parts petioles expressed maximum activity. Maximum NRA was recorded in physiologically mature cotyledonary leaves followed by first and second trifoliates in that order.