Tartrate-resistant acid phosphatase (TRAP) in medullary bone matrix and bone cells was histochemically examined employing estrogen-induced medullary bone of male Japanese quail. Within 2 days after estrogen administration, medullary bone matrix was identified and the matrix and osteoblasts were negative for TRAP activity. Osteoclasts showing TRAP activity were occasionally seen. By 4 days, the projection of medullary bone trabeculae was seen and TRAP became positive in the deeper part of the bone matrix of trabeculae as well as the matrix located near cortical bone, but the surface areas and extending margins of medullary bone trabeculae were negative in activity. Osteoblasts were also negative for TRAP activity. By 7 days, TRAP-positive bone matrix conspicuously increased accompanying increment of bone volume, but TRAP-negative bone matrix was still seen at the surface areas or extending areas of the medullary bone trabeculae. Osteoblasts showed no reaction product. Osteoclasts showing TRAP activity increased in number. The results demonstrate that TRAP accumulates in medullary bone matrix with bone formation. Circumstantial evidence suggests that medullary bone osteoblasts may be involved in TRAP accumulation in the matrix despite their negative activity.