Heterotrimeric G(o) is an abundant brain protein required for negatively reinforced short-term associative olfactory memory in "Drosophila". G(o) is the only known substrate of the S1 subunit of pertussis toxin (PTX) in fly, and acute expression of PTX within the mushroom body neurons (MB) induces a reversible deficit in associative olfactory memory. We demonstrate here that the induction of PTX within the alpha/beta and gamma lobe MB neurons leads to impaired memory acquisition without affecting memory stability. The induction of PTX within these MB neurons also leads to a significant defect in an optimized positively reinforced short-term memory paradigm; however, this PTX-induced learning deficit is noticeably less severe than found with the negatively reinforced paradigm. Both negatively and positively reinforced memory phenotypes are rescued by the constitutive expression of G(o)alpha transgenes bearing the Cys[subscript 351]Ile mutation. Since this mutation renders the G(o) molecule insensitive to PTX, the results isolate the effect of PTX on both forms of olfactory associative learning to the inhibition of the G(o) activation.