Protein splicing involves the excision of an intervening polypeptide, the intein, from flanking polypeptides, the exteins, concomitant with the specific ligation of the exteins. The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be over-expressed and purified as an unspliced precursor, which allows for a detailed, in vitro kinetic analysis of the individual steps of protein splicing. The first order rate constant for splicing of this intein, which has a non-canonical Gln at its C-terminus, is 9.3 x 10^-6 s^-1 at 60°C. The rate constant for splicing increases three-fold with substitution of Asn for the C-terminal Gln. The pseudo first order rate constant of DTT-dependent N-terminal cleavage is 1 x 10^-4 s^-1. The first order rate constant of C-terminal cleavage is 1.2 x 10^-5 s^-1 with Gln at the C-terminal position, 2.8 x 10^-4 s^-1 with Asn, and decreases significantly with mutation of the penultimate His of the intein to Ala. N-terminal cleavage is most efficient between pH 7 and 7.5, and decreases at both more acidic and alkaline pH values, whereas C-terminal cleavage and splicing are both efficient over a broader range of pH values.