Previous work has shown that binding of the TATA box binding protein (TBP) to the TATA box is a rate-limiting step during pre-initiation complex (PIC) formation. Although the transcription of eukaryotic genes normally proceeds in one direction, studies in solution have shown that TBP lacks the information necessary to orient itself on the TATA box. Instead, yeast TBP binds TATA-containing promoters in two orientations that are related by a 180 rotation about TBP's pseudo-2-fold symmetry axis. Recruitment of PIC components by gene-specific activators is considered a primary mechanism of transcriptional enhancement. Here we ask whether activators might function, at least in part, by increasing the fraction of PICs assembled with TBP bound in the orientation necessary for transcription. We use DNA affinity cleavage and a TBP-phenanthroline-copper conjugate to monitor the orientation of TBP in the presence of the well-studied activators Gal4-VP16 and Gal4-AH. In the absence of a transcriptional activator, only 51% of the TBP x TATA box complexes were bound in the orientation necessary for the initiation of transcription. However, in the presence of saturating Gal4-VP16, 87% of the TBP bound to the TATA box was oriented correctly at equilibrium. This increase in orientational specificity corresponds to a free energy difference (Delta Delta G(obs)) of 1.1 kcal x mol(-1) and was accompanied by a dramatic increase in axial specificity, reminiscent of the effects of transcription factors TFIIB and TFIIA reported previously. Gal4-AH also enhanced the orientational and axial specificity of the TBP x TATA complex, although to a lesser extent. We suggest that these effects on specificity represent a variation of recruitment, since they require direct interactions between the activator and a PIC component but only increase the effective concentration of the correctly oriented PIC component. These findings add to increasing evidence that recruitment may encompass a broad range of mechanisms.