Abstract Competitive labelling with tritiated 1-fluoro-2,4-dinitrobenzene was used to determine the ionization constants and reactivities of the two histidines of chymotrypsinogen. The p K a (app.) of histidine-40 is shifted from 8·0 to 6·8 on activation, and its reactivity is decreased by a factor of approximately two. The p K a (app.) of histidine-57 is shifted from 7·5 to 7·0 on activation, and its reactivity is increased by a factor of approximately three. In α-chymotrypsin the pH-reactivity profiles for both histidines are biphasic. In chymotrypsinogen the profile for histidine-40 is biphasic but that for histidine-57 is not. The sharp breaks in the pH-reactivity profiles are attributed to disruption of hydrogen bonding at alkaline pH, except in the case of histidine-57 of chymotrypsinogen, for which the hydrogen bond with aspartic-102 is not easily broken. It is proposed that breaking of this hydrogen bond is an essential step in the catalytic process, and that chymotrypsinogen is prevented from undergoing this step. On hydrolysis of the arginine-15—isoleucine-16 peptide bond, the structural restriction to breaking the hydrogen bond is removed and chymotrypsinogen is activated.