Chymotrypsin and subtilisin BPN' can be inhibited by bis(4-nitrophenyl) methylphosphonate (NMN) and bis(4-nitrophenyl) propylphosphonate (NPN) very efficiently with second-order rate constants, k(i)/K-i, between 544 and 4300 M-1 s(-1) at 25.0 +/-0.1 degrees C at the pH maxima. The second-order rate constants for the inhibition of trypsin are 26.3 +/- 1.4 M-1 s(-1) with NMN and 891 +/- 14 M-1 s(-1) with NPN at pH 8.3 and 25.0 +/-0.1 degrees C. A second stoichiometric equivalent 4-nitrophenol is also lost from 4-nitrophenyl alkylphosphonyl adducts of chymotrypsin but not from trypsin and subtilisin BPN'. Elimination of 4-nitrophenol from the propylphosphonyl adduct is at a rate only about twice the rate of hydrolysis of a comparable phosphonate diester, whereas 4-nitrophenol is eliminated 270 times faster from the methylphosphonyl adduct of chymotrypsin. The activation enthalpies, in kcal/mol, for 4-nitrophenol elimination from 4-nitrophenyl alkylphosphonylchymotrypsin are 15.0 +/- 1.3 for the propyl derivative, 16.4 +/- 0.5 for the methyl derivative in H2O and 18.0 +/- 0.5 in D2O. The activation entropies, in cal mol(-1) K-1, are -29.7 +/- 2.4 for the propyl derivative, -14.8 +/- 0.5 for the methyl derivative in H2O, and -10.3 +/- 0.3 for the methyl derivative in D2O. Partial solvent isotope effects for the elimination of 4-nitrophenol from 4-nitrophenyl methylphosphonylchymotrypsin give beat fits to two-site proton models: These give primary isotope effects between 1.9 and 2.0 (phi(1) double dagger = 0.52 +/- 0.14 or 0.49 +/- 0.07) for a proton in flight, possibly from the water attacking at phosphorus to the catalytic His, and an alpha-secondary effect of 1.3 (phi(2) double dagger = 0.75 +/- 0.20) or a term for solvent contribution of 1.25 (Phi = 0.80 +/- 0.10). The secondary beta-deuterium isotope effect on the elimination of the second 4-nitrophenol from the adduct of chymotrypsin with NMN-1(3) (1 = h or d) is 0.94 +/- 0.2 possibly of hyperconjugative origin. The occurrence and mechanisms of secondary reactions in phosphonylated serine protease enzymes are markedly different from those in phosphonylated cholinesterases.