In the present studies, we have examined the effect of Sp1 on the activation of the human DNA polymerase beta (beta-pol), a TATA-less promoter. A HeLa cell nuclear extract (NE) based in vitro runoff transcription system of core beta-pol promoter human DNA (pbetaP8) three-step kinetic model of transcription initiation were used to describe the kinetic effect of Sp1. The results showed that distal Sp1-binding sites in the core beta-pol promoter are important for transcriptional activation of the pbetaP8 promoter. A detailed kinetic analysis showed that Sp1 stimulates the activity of the pbetaP8 promoter through distal Sp1-binding sites by increasing the amount of recruitment, instead of stimulating the apparent rate of RPc assembly (k1). There was no significant effect of Sp1 on the apparent rate of open complex (RPo) formation (k2) or on the apparent rate of promoter clearance (k3) of the pbetaP8 promoter. These studies define the kinetic mechanisms by which Sp1 may regulate the rate of transcript formation of the pbetaP8 promoter, and these results may have implications for Sp1 regulation of TATA-less promoters.