Saccharomyces cerevisiae Prp43 is a DEAH-box RNA-dependent ATPase that catalyzes the release of excised lariat intron from the mRNA spliceosome. Previous studies identified mutations in Prp43 motifs I, II, and VI that were lethal in vivo and ablated ATP hydrolysis in vitro. Such Prp43 mutants exerted dominant-negative growth phenotypes when expressed in wild type cells and blocked intron release in vitro when added to yeast splicing extracts. Here, we assessed the effects of alanine and conservative substitutions at conserved residues in motifs Ia ((146)TQPRRVAA(153)), IV ((307)LLFLTG(312)), and V ((376)TNIAETSLT(384)) and thereby identified Arg150 (motif Ia), Phe309 (motif IV), Thr376, Leu383, and Thr384 (motif V) as being important for Prp43 function in vivo. Motif V mutations T376V, T384A, and T384V were lethal and dominant negative in vivo, and the mutant proteins inhibited lariat release in vitro. The T384A and T384V proteins were proficient for ATP hydrolysis, suggesting that ATPase activity is necessary, but not sufficient, for Prp43 function. We report that Prp43 hydrolyzes all common NTPs and dNTPs and unwinds short 5'/3' tailed RNA/DNA duplexes in an ATP-dependent fashion. Optimal ATP hydrolysis requires an RNA cofactor of >or=20 nt. Prp43 is largely indifferent to mutations in its C-terminal segment, which is conserved in the DEAH-box splicing factors Prp2, Prp16, and Prp22.