A family of phosphatidylinositol 3-kinases (PI 3-kinase), comprising three major classes (I-III) in terms of substrate specificity and regulation, play important roles in a variety of cell functions. We previously reported that the class-I heterodimeric PI 3-kinase consisting of p110β-catalytic and p85-regulatory subunits is synergistically activated by two different types of membrane receptors, one possessing tyrosine kinase activity and the other activating trimeric G proteins. Here we report an additional unique feature of the p110β/p85 PI 3-kinase. The small GTPase Rab5 was identified as a binding protein for the p110β-catalytic subunit in a yeast two-hybrid screening system. The interaction appears to require at least two separated amino-acid sequences present specifically in the β isoform of p110 and the GTP-bound form of Rab5. The expressions of constitutively active and dominant negative mutants of Rab5 in THP-1 cells induce the stimulation and inhibition, respectively, of protein kinase B activity, which is dependent on the PI 3-kinase product phosphatidylinositol 3, 4, 5-triphosphate. These results suggest that there is a specific interaction between GTP-bound Rab5 and the p110β/p85 PI 3-kinase, leading to efficient coupling of the lipid kinase product to its downstream target, protein kinase B.