We hypothesized that the cytoskeletal network in vascular smooth muscle cells (VSMC) is critical to the signaling pathways from angiotensin (ANG) II-receptor subtype 1 (AT₁) activation to intracellular Ca²⁺ (Ca²⁺_i) release from internal stores and Ca²⁺ influx. This was tested in spontaneously hypertensive rats (SHR), in which differences were reported in cultured aortic VSMC Ca²⁺_i regulation and G protein function compared with those in normotensive Wistar-Kyoto (WKY) rats. In cultured aortic VSMC, disorganization of actin filaments with cytochalasin D (2 µmol/l) decreased the ANG II-induced Ca²⁺_i release from internal stores and the ANG II-induced Ca²⁺ influx in SHR in a reversible fashion, whereas it was without effect in WKY rats. On the other hand, blocking the dynamic state of the microtubule network significantly reduced ANG II-induced Ca²⁺_i release from internal stores but was without effect on Ca²⁺ influx in either SHR or WKY rats. This study demonstrates for the first time that, in the SHR, actin filaments play a major role in linking AT₁-receptor activation to both Ca²⁺_i release mechanisms and capacitative Ca²⁺ influx. Furthermore, a functionally intact microtubule system is a necessary prerequisite for ANG II-induced Ca²⁺_i release in both strains.