NaeI endonuclease must bind two DNA sites for cleavage to occur. NaeI was purified to apparent homogeneity and used to determine the rate-limiting step for DNA cleavage and to measure NaeI's specificity for its cognate recognition site. Steady-state cleavage by NaeI in the presence of effector DNA (activated) gave values of 0.045 s-1 and 10 nM for kcat and KM for M13 DNA substrate, respectively, but values of 0.4 s-1 and 170 nM, respectively, for an M13 DNA fragment substrate. Single-turnover cleavage of M13 DNA demonstrated that DNA strand scission is not rate-limiting for turnover of NaeI. Transient kinetic analysis of M13 DNA cleavage by NaeI showed an initial burst of substrate cleavage that was proportional to NaeI concentration, implying that product release is rate-limiting for turnover of NaeI. The NaeI effector and substrate binding sites were found to prefer cognate over noncognate sequences by 10(3)-fold and at least 40-500-fold, respectively. kcat for noncognate recognition sequence was at least 10(6)-fold lower than that for cognate. The specificity of activated NaeI, as measured by kcat/KM, for noncognate recognition sequence was 10(8)-fold lower than that for cognate, and over 10(11)-fold lower when the decreased affinity for noncognate sequence at the effector binding site was taken into account. This specificity is approximately 10(4)-fold larger than for any other restriction enzyme measured.