When muscle fibers contract, blood flow requirements increase along their entire length. However, the organization of capillary perfusion along muscle fibers is unclear. The microvascular unit (MVU) is defined as a terminal arteriole and the group of capillaries it supplies. We investigated whether neighboring MVUs along the fiber axis perfused the same group of muscle fibers by using the parallel-fibered retractor muscle. Hamsters were anesthetized and perfused with Microfil to visualize MVUs relative to muscle fibers. Fields of study, which encompassed five to seven neighboring MVUs along a muscle fiber, were chosen from the interior of muscles and along muscle edges. On average, MVUs were 1 mm in length, 0.50 mm in width, and 0.1 mm deep; segments of approximately 30 fibers were contained in this tissue volume of 0.05 mm3 (20 MVUs/mg muscle). The total distance across muscle fibers encompassed by a pair of MVUs is designated "union" (U); the fraction of this distance common to both MVUs is designated "intersection" (I). The ratio of I to U for the widths of neighboring MVUs provides an index of MVU alignment along muscle fibers (e.g., I/U = 1.0 indicates complete alignment, where the fibers perfused by one MVU are the same as those perfused by the neighboring MVU). We found that I/U along muscle edges (0.71 +/- 0.02) was greater (P < 0.05) than the ratio measured within muscles (0.66 +/- 0.02). A model predicted a maximum I/U of 0.58 with random MVU alignment. Thus measured values were closer to random than to complete alignment. These findings indicate that an increase in blood flow along muscle fibers requires the perfusion of many MVUs and imply that vasodilation is coordinated among the parent arterioles from which corresponding MVUs arise.