The ability of various kinds of RNA molecules to activate the ATP hydrolysis reaction catalyzed by the p transcription termination factor from Escherichia coli has been studied. The most active RNA polymers are those containing cytidylate residues and very little ordered structure. Free poly(C) is the most active homopolymer; it is 45 times more active than poly(U), which is the only non-cytidine containing RNA that has detectable activity. Poly(C) has no activity when complexed with poly(I) or when the chain lengths are shorter than 22 nucleotides long. Although cytidylate residues are important they need not be frequent; a random copolymer of uridine and cytidine nucleotides with as few as 1 cytidylate residue out of 20 is as active as poly(C). The extent of activation with poly(C) depends on the ratio of p to poly(C). Poly(C) becomes saturated with p at a ratio of 1.8 ng of p/pmol poly(C), which is equivalent to one p monomer/27 nucleotides. A further increase in this ratio leads to a reduction in p activity. Decreasing the length of the poly(C) does not alter the observed saturation value but does decrease the rate of ATP hydrolysis when the RNA is in excess. The possible relevance of these results to p termination activity is discussed.