Protein-splicing inteins are widespread in nature and have found many applications in protein research and engineering. The mechanism of protein splicing typically requires a nucleophilic amino acid residue at both position 1 (first residue of intein) and position +1 (first residue after intein), however it was not clear whether or how the three different nucleophilic residues (Cys, Ser, and Thr) would work differently at these two positions. To use intein in a target protein of interest, one needs to choose an intein insertion site to have a nucleophilic residue at position +1, therefore it is desirable to know what nucleophilic residue(s) are preferred by different inteins. In this study we began with a statistical analysis of known inteins, which showed an unequal distribution of the three nucleophilic residues at positions 1 and +1, and then subjected six different mini-inteins to site-directed mutagenesis to systematically test the functionality of the three nucleophilic residues at the two positions. At position 1, most natural inteins had Cys and none had Thr. When the Cys at position 1 of the six inteins was mutated to Ser and Thr, the splicing activity was abolished in all except one case. At position +1, Cys and Ser were nearly equally abundant in natural inteins, and they were found to be functionally interchangeable in the six inteins of this study. When the two positions were studied as 1/+1 combination, the Cys/Ser combination was abundant in natural inteins, whereas the Ser/Cys combination was conspicuously absent. Similarly, all of the six inteins of this study spliced with the Cys/Ser combination, whereas none spliced with the Ser/Cys combination. These findings have interesting implications on the mechanism of splicing and the selection of intein insertion sites, and they also produced two rare mini-inteins that could splice with Thr at position +1.