The pbs3-1 mutant, identified in a screen for Arabidopsis (Arabidopsis thaliana) mutants exhibiting enhanced susceptibility to the avirulent Pseudomonas syringae pathogen DC3000 (avrPphB), also exhibits enhanced susceptibility to virulent P. syringae strains, suggesting it may impact basal disease resistance. Because induced salicylic acid (SA) is a critical mediator of basal resistance responses, free and glucose-conjugated SA levels were measured and expression of the SA-dependent pathogenesis-related (PR) marker, PR1, was assessed. Surprisingly, whereas accumulation of the SA glucoside and expression of PR1 were dramatically reduced in the pbs3-1 mutant in response to P. syringae (avrRpt2) infection, free SA was elevated. However, in response to exogenous SA, the conversion of free SA to SA glucoside and the induced expression of PR1 were similar in pbs3-1 and wild-type plants. Through positional cloning, complementation, and sequencing, we determined that the pbs3-1 mutant contains two point mutations in the C-terminal region of the protein encoded by At5g13320, resulting in nonconserved amino acid changes in highly conserved residues. Additional analyses with Arabidopsis containing T-DNA insertion (pbs3-2) and transposon insertion (pbs3-3) mutations in At5g13320 confirmed our findings with pbs3-1. PBS3 (also referred to as GH3.12) is a member of the GH3 family of acyl-adenylate/thioester-forming enzymes. Characterized GH3 family members, such as JAR1, act as phytohormone-amino acid synthetases. Thus, our results suggest that amino acid conjugation plays a critical role in SA metabolism and induced defense responses, with PBS3 acting upstream of SA, directly on SA, or on a competitive inhibitor of SA.