We measured membrane fluidity and fatty acid composition in cultured vascular smooth muscle cells from stroke-prone spontaneously hypertensive and Wistar-Kyoto normotensive rats. Membrane fluidity was measured as a lateral diffusion of 5 N (octadecanoyl) aminofluorescein using fluorescence microscopy and fluorescence recovery after photobleaching. Fatty acid composition of membrane fractions was measured using high performance liquid chromatography. Lateral diffusion was significantly lower (the membrane had lower fluidity) in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats as compared to those cells isolated from Wistar-Kyoto reference strain. The ratio of arachidonic acid to total fatty acids was 0.058 +/- 0.007 in the plasma membrane from stroke-prone spontaneously hypertensive rats and 0.036 +/- 0.005 in that from Wistar-Kyoto rats, P = .005. Similarly, the ratios of arachidonic to oleic acid and arachidonic to palmitic acid were significantly greater in cells from stroke-prone spontaneously hypertensive rats (P = .002 for difference in each ratio). These results show decreased lateral diffusion (decreased membrane fluidity) in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats. This is associated with increased content of arachidonic acid, the major precursor of prostaglandins and other eicosanoids. We postulate that local changes in the unsaturated fatty acid composition related to arachidonic acid storage and release contribute to reduced membrane fluidity in stroke-prone spontaneously hypertensive rats.