ABSTRACT: We investigated changes in connective tissue components of masseter (MA) muscle in Japanese black heifers (n= 6) in concentrate- and roughage-fed groups (groups C and R, respectively). Body weight, at slaughter, of experimental heifers in group C (272.3 ± 22.3 kg) was higher (P < 0.05) than that of group R (213.8 ± 27.5 kg). However, muscle weight and myofiber diameter (superficial and deep layers) of MA muscle did not differ between groups C and R. In contrast, total mastication duration of group R was longer (P < 0.05) than that of group C. MA muscle of groups C and R was composed only of type I myofiber. Using immunohistochemical/confocal laser-scanning microscopy, type I collagen was observed mainly in perimysium, and type V and VI collagen were observed in perimysium and endomysium of both groups. Type IV collagen and laminin were observed only in the endomysium in both groups. However, type III collagen and fibronectin were strongly apparent in the perimysium and endomysium in group R. Connective tissue components in the perimysium of groups C and R were observed to form plate-shaped layers. On the other hand, honeycomb-shaped connective tissue components were seen in the endomysium-surrounded muscle fibers. In particular, fibronectin was strongly observed in the perimysium and endomysium in group R. These results indicate that there are different developmental changes among connective tissue components in MA muscle in response to mastication. The immunohistochemical/confocal laser-scanning microscopic method is useful to investigate the structural relationship among connective tissue components in skeletal muscle.