The increased capillary fluid filtration required to create a rapid edema formation in acute inflammation can be generated by lowering the interstitial fluid pressure (P_IF). The lowering of P_IF appears to involve dynamic ₁-integrin-mediated interactions between dermal cells and extracellular matrix fibers. The present study specifically investigates the role of the cell cytoskeleton, i.e., the contractile apparatus of cells, in controlling P_IF in rat skin as the integrins are linked to both the cytoskeleton and the extracellular matrix. P_IF was measured using a micropuncture technique in the dorsal skin of the hind paw at a depth of 0.2-0.5 mm and following the induction of circulatory arrest with the intravenous injection of KCl in pentobarbital anesthesia. This procedure prevented the transcapillary flux of fluid and protein leading to edema formation in acute inflammation, which in turn can increase the P_IF and therefore potentially mask a decrease of P_IF. Control P_IF (n = 42) averaged 0.8 ± 0.5 (means ± SD) mmHg. In the first group of experiments, subdermal injection of 2 µl cytochalasin D, a microfilament-disrupting drug, lowered P_IF to an average of 2.8 ± 0.7 mmHg within 40 min postinjection (P < 0.05 compared with control). Subdermal injection of vehicle (10% DMSO in PBS or PBS alone) did not change the P_IF (P > 0.05). Lowering of the P_IF was not observed after the injection of colchicine or nocodazole, which specifically disrupts microtubuli in cultured cells. In the second group of experiments, 2 µl of cytochalasin D injected subdermally into rats with intact circulation increased the total tissue water (TTW) and albumin extravasation rate (E_ALB) by 0.7 ± 0.2 and 0.4 ± 0.3 ml/g dry wt, respectively (P < 0.05 compared with vehicle). Nocodazole and colchicine did not significantly alter the TTW or E_ALB compared with the vehicle (P > 0.05). Taken together, these findings strongly suggest that the connective tissue cells can participate in control of P_IF via the actin filament system. In addition, the observation that subdermal injection of cytochalasin D lowered P_IF indicates that a dynamic assembly and disassembly of actin filaments also occurs in the cells of dermal tissues in vivo.