The most active factor VIIa (FVIIa) variants identified to date carry concurrent substitutions at positions 158, 296 and 298 with the intention of generating a thrombin-mimicking motif, optionally combined with additional replacements within the protease domain [Persson, E., Kjalke, M. & Olsen, O. H. (2001) Proc. Natl Acad. Sci. USA98, 13583-13588]. Here we have characterized variants of FVIIa mutated at one or two of these positions to assess the relative importance of the individual replacements. The E296V and M298Q mutations gave an increased intrinsic amidolytic activity (about two- and 3.5-fold, respectively) compared with wild-type FVIIa. An additive effect was observed upon their combination, resulting in the amidolytic activity of E296V/M298Q-FVIIa being close to that of the triple mutant. The level of amidolytic activity of a variant was correlated with the rate of inhibition by antithrombin (AT). Compared with wild-type FVIIa, the Ca2+ dependence of the intrinsic amidolytic activity was significantly attenuated upon introduction of the E296V mutation, but the effect was most pronounced in the triple mutant. Enhancement of the proteolytic activity requires substitution of Gln for Met298. The simultaneous presence of the V158D, E296V and M298Q mutations gives the highest intrinsic activity and is essential to achieve a dramatically higher relative increase in the proteolytic activity than that in the amidolytic activity. The N-terminal Ile153 is most efficiently buried in V158D/E296V/M298Q-FVIIa, but is less available for chemical modification also in the presence of the E296V or M298Q mutation alone. In summary, E296V and M298Q enhance the amidolytic activity and facilitate salt bridge formation between the N-terminus and Asp343, E296V reduces the Ca2+ dependence, M298Q is required for increased factor X (FX) activation, and the simultaneous presence of the V158D, E296V and M298Q mutations gives the most profound effect on all these parameters.