Class I phosphoinositide 3-kinase (PI3K) enzymes produce lipid second messengers that regulate diverse biological processes. In neutrophils, activation of class I PI3Ks is crucial for directed motility. However, the roles of class I PI3K isoforms in the basal and directed motility of lymphocytes are poorly understood. Using two-photon microscopy, we have identified PI3K as an important signaling molecule for maintaining basal T and B lymphocyte motility and homing in the intact lymph node. Pharmacological inhibition of PI3K catalytic isoforms exerted broad effects on basal lymphocyte motility, including changes in homing kinetics, localization of B cells within the lymph node, and reduced cell velocities. Lymphocytes deficient in either or both of the Class IA PI3K regulatory subunits p85α and p85β also exhibited reduced velocities, to varying extents depending on cell-type and isoform-specificity. B cells deficient in p85α exhibited gross morphological abnormalities, which were not evident in cells treated with a PI3K inhibitor. Our results show, for the first time, that class IA PI3Ks play an important role in maintaining basal lymphocyte motility, and that p85α regulatory subunit expression is required to maintain B cell morphology in a manner independent of PI3K catalytic function. Moreover, we demonstrate distinct roles for catalytic domain function and class IA PI3K regulatory domain activity in lymphocyte motility, homing and homeostatic localization of resting B cells.