The effects of Mg++, Mn++, and KCl addition, individually and in combination, on the rate of DNA- and RNA-primed DNA synthesis by avian myeloblastosis virus DNA polymerase (reverse transcriptase) using a variety of natural and synthetic template-primer combinations were examined. Optimal divalent cation concentrations were found to vary by as much as 10-fold depending upon the template-primer used to direct synthesis. Addition of KCl to reaction mixtures containing optimal divalent cation concentrations produced stimulation or inhibition of DNA synthesis which was also template-specific. DNA synthesis on the modified template poly (2'-0-methylcytidylate) was uniquely stimulated by combinations of divalent cations. With Mg++ as divalent cation, deviations from classical Michaelis-Menten kinetics of substrate saturation were observed with all template-primers tested.