A specific tumour necrosis factor alpha ribozyme (TNF-alpha-Rz) binding activity has been purified and identified by N-terminal microsequencing as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The purified protein as well as commercial GAPDH binds tightly to TNF-alpha ribozyme compared to a variety of other ribozymes and RNAs. Binding of GAPDH to the TNF-alpha-Rz and its derivatives was inhibited by NAD+ and ATP, suggesting that the GAPDH Rossmann fold structure is a part of the ribozyme binding site. Interestingly, GAPDH increased the in vitro cleavage rates of hammerhead ribozymes by up to 25-fold, while no significant stimulation was observed with the lactate dehydrogenase (LDH). This effect was found to be due to the unfolding activity of GAPDH. In fact, pulse-chase experiments demonstrate directly that GAPDH has the capacity to accelerate the ribozyme/substrate association, especially of ribozymes and/or substrates whose predicted secondary structure might interfere with the association step. Under our conditions, the presumed unfolding activity of GAPDH also enhances the turnover of ribozymes by increasing the rate of product dissociation, although only for short cleavage products. Longer duplexes required more incubation time to dissociate. In vitro non-specific interaction of the GAPDH with hammerhead ribozymes and RNA substrates was found to be adequate for the cleavage enhancement effect to occur. However, an analysis of the ability of various prototypical ribozymes to inhibit the expression of interleukin-2 suggests that the addition of a sequence having a high affinity for GAPDH improves the efficacy of ribozymes in the cells. Thus the characterization of cellular proteins with unfolding activity, which specifically bind to hammerhead ribozyme, should facilitate the design of a more effective ribozyme in vivo.