Guidance and monitoring of high intensity focused ultrasound (HIFU) therapy, using ultrasound imaging, has primarily utilized formation of a hyperechoic region at the HIFU focus. We investigated biologic and physical mechanisms of a hyperecho, as well as safety of this phenomenon, using thermal, acoustic and light microscopy observations. Single, short-duration HIFU pulses (30-60 ms) were able to produce a hyperechoic region at the HIFU focus, 2 cm deep in a rabbit thigh muscle. When hyperechoic regions appeared, inertial cavitation was detected in vivo using a custom-made passive cavitation detection system. Light micrographs showed a large number of cavities (approximately 100/mm3), 1-10 microm in diameter, in a cytoplasm of cells located at the HIFU focus. Blood congestion was observed around a focal region, indicating an injury of microvasculature. Cellular necrosis was observed at 2 d after the treatment, while healing, scar tissue formation and regeneration were observed at 7 d and 14 d. The results indicate that a possibility of adverse tissue effects has to be taken into consideration when the hyperecho formation, induced by very-short HIFU pulses, is used for pretreatment targeting.