In these studies we have examined rat kidneys biochemically and microscopically to determine where myosin I beta is located before, during, and after an acute ischemic injury. Myosin I beta is present in multiple tubule segments including the brush border (BB) of the proximal tubule cell (PTC). Its distribution is severely altered by a 15-min renal artery clamp. Myosin I beta is present in the urine during reflow and is found in the numerous cellular blebs arising from the damaged PTC and other tubules. Two hours of reflow result in a decrease in BB myosin I beta staining and an increase in its cytoplasmic staining. Interestingly, the return of the F-actin in the BB precedes the return of the myosin I beta, suggesting that this myosin I isoform may not play a role in rebuilding the microvilli after an ischemic injury. A nonstructural role for this myosin, such as transport or channel regulation, is supported by its presence in many tubule segments, all of which have transport and channel requirements but do not all contain microvilli.