Many membrane transport systems are altered by changes in the state of the actin cytoskeleton. Although an intact microtubule network is required for hypertonic activation of the betaine transporter (BGT1), the possible role of the actin cytoskeleton is unknown. BGT1 function in Madin-Darby canine kidney cell monolayers was assessed as Na(+)-dependent uptake of GABA, following disassembly of F-actin by cytochalasin D (1.0 microM) or latrunculin A (0.6 microM). Both drugs significantly increased (P < 0.001) the activation of BGT1 transport by 24-h hypertonicity (500 mosmol/kgH(2)O). In contrast, the hypertonic upregulation of Na(+)-dependent alanine uptake remained unaltered by cytochalasin D. Disruption of F-actin did not interfere with downregulation of BGT1 transport when cells were transferred from hypertonic to isotonic medium. Immunofluorescence staining revealed colocalization of BGT1 and F-actin at the plasma membrane of hypertonic cells. Surface biotinylation revealed no major change in BGT1 protein abundance after cytochalasin D action, suggesting that stimulation of hypertonic activation of BGT1 transport is due to increased activity of existing BGT1 transporters.