Human PrimPol binds DNA with a 34-fold higher affinity with Mn2+ than with Mg2+. PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn2+ than with Mg2+. The substitution fidelity of PrimPol is 100–1000 fold lower with Mn2+ than with Mg2+. The sugar selectivity of PrimPol is 57–1800 with Mn2+ and 150–4500 with Mg2+. PrimPol incorporates cytarabine and gemcitabine, but not emtricitabine and lamivudine.