Gene expression is regulated by a large complex of proteins that bind to the promoter/enhancer region of a gene. We determined whether a streptavidin-bead binding assay might be useful in detecting individual proteins in the complex comprising transactivators, coactivators, mediators, and general transcription factors. We used biotinylated cyclooxygenase-2 promoter probes as a model. Nuclear extracts obtained from human fibroblasts treated with or without an agonist were incubated with a 5(')-biotinylated probe and streptavidin-agarose beads at room temperature for 1h. After centrifugation, the pellet was washed and proteins in the complex were assessed by immunoblots. An array of transcription factors was detectable concurrently in the same batch of pellets at basal state. p300 and its associated factor PCAF levels but not Srb7, Med7, or TFII(B) were increased by phorbol ester or tumor necrosis factor alpha stimulation. Only trace of CREB-binding protein was detected. These results suggest that p300 and PCAF are the predominant coactivators for COX-2 promoter activation. Our findings indicate that the streptavidin-bead pulldown assay is valuable for determining the binding of a large number of transcription factors to promoter/enhancer and evaluating the relationship of protein binding with regulation of gene expression.