Previous studies indicated that the determinants of coenzyme specificity in ferredoxin-NADP+ reductase (FNR) from Anabaena are situated in the 2'-phosphate (2'-P) NADP+ binding region, and also suggested that other regions must undergo structural rearrangements of the protein backbone during coenzyme binding. Among the residues involved in such specificity could be those located in regions where interaction with the pyrophosphate group of the coenzyme takes place, namely loops 155-160 and 261-268 in Anabaena FNR. In order to learn more about the coenzyme specificity determinants, and to better define the structural basis of coenzyme binding, mutations in the pyrophosphate and 2'-P binding regions of FNR have been introduced. Modification of the pyrophosphate binding region, involving residues Thr-155, Ala-160, and Leu-263, indicates that this region is involved in determining coenzyme specificity and that selected alterations of these positions produce FNR enzymes that are able to bind NAD+. Thus, our results suggest that slightly different structural rearrangements of the backbone chain in the pyrophosphate binding region might determine FNR specificity for the coenzyme. Combined mutations at the 2'-P binding region, involving residues Ser-223, Arg-224, Arg-233, and Tyr-235, in combination with the residues mentioned above in the pyrophosphate binding region have also been carried out in an attempt to increase the FNR affinity for NAD+/H. However, in most cases the analyzed mutants lost the ability for NADP+/H binding and electron transfer, and no major improvements were observed with regard to the efficiency of the reactions with NAD+/H. Therefore, our results confirm that determinants for coenzyme specificity in FNR are also situated in the pyrophosphate binding region and not only in the 2'-P binding region. Such observations also suggest that other regions of the protein, yet to be identified, might also be involved in this process.