S-RNase is associated with the gametophytic self-incompatibility of flowering plants in Solanaceae and, on the basis of sequence homology, belongs to the RNase T2 family. To identify the active site residues in S-RNase, Nicotiana alata S6-RNase was studied by chemical modification. S6-RNase was inactivated with iodoacetic acid under conditions similar to those used for the inactivation of RNase T2. No inactivation took place in the presence of 2'GMP. Analysis of carboxymethylated S6-RNase revealed that the S-carboxymethylation of Cys95 caused inactivation of the enzyme and that the two histidine residues corresponding to two essential histidine residues of RNase T2 remained intact. Treatment of S6-RNase with diethyl pyrocarbonate (DEPC) resulted in loss of enzyme activity, and the enzyme was protected from inactivation in the presence of 2'GMP. The ethoxycarbonylated residues in DEPC-inactivated S6-RNase were analyzed by mass spectrometry, which also provided structural information on sugar moieties attached to Asn27 and Asn37. His31 was modified with DEPC in the absence of 2'GMP and was not modified in its presence. His31 and His91 are conserved in all members of the RNase T2 family sequenced so far, but Cys95 is not conserved in all known Solanaceae S-RNases. These results suggest that His31, possibly together with His91, corresponding to His115 at the active site of RNase T2, is essential to the function of S6-RNase, but Cys95 is not essential though its S-carboxymethylation causes inactivation.