Soil N is known to affect N2 fixation in legumes. Increased cowpea yields might be achieved by increasing N assimilation through greater symbiotic fixation, but identification of high‐fixing genotypes requires an effective screening technique. The purpose of this investigation was to determine the optimum level of supplemental N for maximum N2 fixation in cowpeas grown under greenhouse and field conditions, and the relative contributions of the measured variables on nitrogenase activity. Three cowpea cultivars were tested for N2 fixation, N2 (C2H2), under greenhouse conditions with 0, 5, 10, and 20 ppm N applied as Ca(NO3)2, and under field conditions with 0, 11.2, 22.4, and 44.8 kg/ha N applied as Ca(NO3)2. The soil was Vertic Albaqualf, fine, Montmorillonitic thermic. Nitrogenase activity decreased with increasing rates of N. Path analysis indicated that nitrate effects on N2 fixation activity were primarily on nodule mass, with the inhibition of nodule growth rather than nodule initiation. Nodule number was not a reliable predictor of N2 fixation potential of cowpea genotypes and was negatively correlated with nitrogenase activity. The results clearly indicated no advantage of applied nitrate in promoting maximum genotypic differences for N2 fixation.