Biosynthetic thiolase from Zoogloea ramigera was inactivated with a mechanism-based inactivator, 3-pentynoyl-S-pantetheine-11-pivalate (3-pentynoyl-SPP) where K1 = 1.25 mM and kinact = 0.26 min-1, 2,3-pentadienoyl-SPP obtained from nonenzymatic rearrangement of 3-pentynoyl-SPP where K1 = 1.54 mM and kinact = 1.9 min-1 and an affinity labeling reagent, acryl-SPP. The results obtained with the alkynoyl and allenoyl inactivators are taken as evidence that thiolase from Z. ramigera is able to catalyze proton abstraction uncoupled from carbon-carbon bond formation. The inactivator, 3-pentynoyl-SPP and the affinity labeling reagent, acryl-SPP, trap the same active site cysteine residue, Cys-378. To assess if Cys-378 is the active site residue involved in deprotonation of the second molecule of acetyl-CoA, a Gly-378 mutant enzyme was studied. In the thiolysis direction the Gly-378 mutant was more than 50,000-fold slower than wild type and over 100,000-fold slower in the condensation direction. However, the mutant enzyme was still capable of forming the acetyl-enzyme intermediate and incorporated 0.81 equivalents of 14C-label after incubation with [14C]Ac-CoA for 60 min. The reversible exchange of 32P-label from [32P]CoASH into Ac-CoA, catalyzed by the Gly-378 mutant enzyme, proceeded with a Vmax (exchange) 8,000-fold less than the wild type enzyme but at least 10-fold faster than the overall condensation reaction. These data provide evidence that Cys-378 is the active site base.