The time course of removal of thymine by thymine DNA glycosylase has been measured in vitro. Each molecule of thymine DNA glycosylase removes only one molecule of thymine from DNA containing a G.T mismatch because it binds tightly to the apurinic DNA site left after removal of thymine. The 5'-flanking base pair to G.T mismatches influences the rate of removal of thymine: kcat values with C.G, T.A, G.C, and A.T as the 5'-base pair were 0.91, 0.023, 0. 0046, and 0.0013 min-1, respectively. Thymine DNA glycosylase can also remove thymine from mismatches with S6-methylthioguanine, but, unlike G.T mismatches, a 5'-C.G does not have a striking effect on the rate: kcat values for removal of thymine from SMeG.T with C.G, T. A, G.C, and A.T as the 5'-base pair were 0.026, 0.018, 0.0017, and 0. 0010 min-1, respectively. Thymine removal is fastest when it is from a G.T mismatch with a 5'-flanking C.G pair, suggesting that the rapid reaction of this substrate involves contacts between the enzyme and oxygen 6 or the N-1 hydrogen of the mismatched guanine as well as the 5'-flanking C.G pair. Disrupting either of these sets of contacts (i.e. replacing the 5'-flanking C.G base pair with a T.A or replacing the G.T mismatch with SMeG.T) has essentially the same effect on rate as disrupting both sets (i.e. replacing CpG.T with TpSMeG.T), and so these contacts are probably cooperative. The glycosylase removes uracil from G.U, C.U, and T.U base pairs faster than it removes thymine from G.T. It can even remove uracil from A.U base pairs, although at a very much lower rate. Thus, thymine DNA glycosylase may play a backup role to the more efficient general uracil DNA glycosylase.