We have investigated the structural determinants of the unique functional properties of complement factor D by constructing and testing a series of trypsin-like mutants of the enzyme. Mutational replacement of the primary substrate-binding pocket of factor D with that of trypsin resulted in a mutant (M1) with greatly reduced proteolytic activity and slightly reduced reactivity toward small thioester substrates. Combining the M1 mutations with substitution of Tyr for Ser94, previously shown to enhance substantially both the proteolytic and esterolytic activities of factor D, produced a mutant (M2) with reactivities similar to M1. Replacement of the surface loop formed by residues 184-188 of M1 and M2 with the corresponding loop of trypsin produced mutants exhibiting one and two orders of magnitude higher esterolytic activity, respectively, than native factor D. However, the proteolytic activity of both mutants was similar to that of M1 and M2. We conclude that loop184-188 is an important determinant of the geometry of the primary specificity pocket of factor D. The low proteolytic activity of these mutants supports the proposal that the proteolytically active conformation of factor D is induced by its natural substrate, C3bB.