This paper describes the consequences of alanine-scanning mutagenesis on 28 positions of the second epidermal growth factor (EGF-2) domain of factor IX. We identified four positions of Gln(97), Phe(98), Tyr(115), and Leu(117) that are critical for secretion of factor IX. Of the remaining mutations, 4 mutants (V86A, E113A, K122A, and S123A) are as active as wild-type factor IX (IXwt); 16 (D85A, K100A, N101A, D104A, N105A, R116A, E119A, T87A, I90A, K91A, R94A, E96A, S102A, K106A, T112A, and N120A) retain reduced but detectable activity, and 4 (N89A, N92A, G93A, and V107A) are nearly inert in the clotting assay. Both factor XIa and the factor VIIa-tissue factor complex effectively catalyzed the activation of these mutants except N89A. The mutant V107A failed to form the factor tenase complex with factor VIIIa because of a 35-fold increase in K(d). The mutants N89A and N92A did not compete with factor IXwt for factor VIIIa binding, and G93A exhibited a 6-fold increase in K(i) values in the competitive binding assay. It appears that mutations at these positions have significantly affected the interaction between factor IX and factor VIIIa, although other mutations had little effect on the binding of factor IX to factor VIIIa. Mutations in two regions, Thr(87)-Gly(93) and Asn(101)-Val(107), significantly increased the K(m) value of factor IXa (2-10-fold) in cleavage of factor X in the absence of factor VIIIa. In the presence of factor VIIIa, the catalytic efficiency of each mutant toward factor X paralleled its clotting activity. Briefly, we propose two relatively distinctive functions of factor IX for two adjacent regions in the EGF-2 domain; the first loop region (residues 89-94) is involved with the binding of its cofactor, factor VIIIa, and the third loop with connected beta-sheets (residues 102-108) is involved in the proper binding to the substrate, factor X.