We have shown that accurate initiation of productive RNA synthesis in vitro at the adenovirus 2 major late promoter is accompanied by abortive initiation of very short transcripts (Luse, D. S., and Jacob, G. A. (1987) J. Biol. Chem. 262, 14990-14997). We made a set of sequence variants of this promoter, using every possible base at position -28 (in the TATA box) in the context of either the normal base (A) or a T at position +1 on the nontemplate strand. All changes from wild type reduced promoter strength. The two weakest promoters were 10- and 30-fold less active than wild type in productive RNA synthesis. We tested the possibility that the down mutations also caused an increase in the proportion of in vitro initiations which are abortive. This effect was seen only with the two weakest members of the promoter set. For these promoters, which share an A----C change at the -28 position of the TATA box, the ratio of abortive to productive initiations was 3-4-fold higher than for the other promoters. Interestingly, the sequence change at +1, although a down mutation, did not lead to an increase in abortive initiation.