Pediatric cancer chemotherapy is known to cause bone growth arrest and osteoporotic changes, and yet the underlying mechanisms remain largely unknown. This project investigated effects of acute chemotherapy with topoisomerase inhibitor etoposide (Eto, 80 mg/kg), alkylating agent cyclophosphamide (Cyc, 240 mg/kg) or their combination (Cyc 120 mg/kg + Eto 50 mg/kg) on structural and cellular changes in the growth plate cartilage and metaphyseal bone, two important regions responsible for bone growth and bone mass accumulation. On day 3 after a single injection with either of the three treatments, although the total growth plate thickness was not significantly altered, the cellularity and height of the proliferative zone were significantly reduced. It was shown that while Eto suppressed chondrocyte proliferation, Cyc induced apoptosis in the growth plate proliferative zone. In the metaphysis, although osteoblastic cell surface was decreased in all three treated groups, the trabecular bone bone volume (BV/TV%) was not significantly altered on day 3. On the other hand, the acute chemotherapy reduced heights of both primary and secondary spongiosa trabecular bone. Therefore, Eto and/ or Cyc chemotherapy altered survival or proliferation of growth plate chondrocytes and metaphyseal osteoblastic cells and reduced heights of metaphyseal spongiosa trabecular bone, which may contribute to chemotherapy side effects of these two drugs on bone lengthening and bone mass accumulation.