This study was performed to measure the acoustic propagation speed in live human aortic smooth-muscle cells (HASMC), using scanning acoustic microscopy (SAM) and a novel measurement theory that permits the measurement of the acoustic propagation speed in biological samples of unknown thickness. C-mode and X-Z-mode images of HASMC under three different conditions: growing (G); differential (D); and on hypotonic loading (H), were acquired using 100-MHz, 450-MHz and 600-MHz ultrasound. The images exhibit features related to the cell surface curvature and intracellular structure. The theory supporting the methodology is derived in this article and makes use of the interference fringes within the focusing lens of the high-frequency transducer. The propagation speed in the cells was calculated from the location of the interference fringe on the C-mode images and the fringe shift on the X-Y-mode images with 450-MHz ultrasound. The propagation speed in D (1624 +/- 16 m/s) was significantly higher than those in G (1571 +/- 14 m/s, p < 0.05) and H (1585 +/- 8 m/s, p < 0.05). Scanning acoustic microscope measurements, along with the described theory, are useful for studying the acoustic properties of live cells ex vivo and have applications in both pathophysiology and biomechanics.