The log of the rate of the chemical step of hammerhead cleavage in Mg2+ increases linearly with pH between pH 5.7 and 8.9. A slope of approximately 1 indicates that a single deprotonation is required for cleavage. Hammerhead pH-rate profiles with Ca2+, Mn2+, Co2+, and Cd2+ correlate well with the pKa's of these ions in water. This relationship between the PKa'S and the pH-rate profile suggests that a metal hydroxide bound to the hammerhead RNA acts as the base in the cleavage mechanism.