It is well recognized that, in the mouse, high-dose estrogen induces sclerosis within the shaft of long bones, an action that is largely thought to reflect increased osteoblastic cellular activity. We undertook to characterize this response in more detail, by performing a histologic analysis of the early changes induced by high-dose estrogen in the tibial cavity of young intact female mice. Female mice were sacrificed immediately before or 4, 8, 12, or 24 days after commencing subcutaneous injections of 17beta-estradiol (500 microg/animal/week), and longitudinal tibial sections were subsequently examined. Estrogen was found to cause a rapid gain in cancellous bone, with cancellous bone volume increasing by approximately 50% after 8 days, and by 5-fold after 24 days. Analysis of cancellous double-labeled surfaces revealed that this gain in bone reflected the emergence of new cancellous bone formation sites within the medullary cavity, rather than the reactivation and extension of formation over pre-existing bone surfaces. Comparison of the time course of these changes between proximal and distal regions of the proximal tibial metaphysis suggested that these new cancellous formation sites appear as a rapid wave extending distally from the secondary spongiosa. Alkaline phosphatase (ALP) immunocytochemistry revealed that, by 12 days after estrogen administration, a population of strongly ALP positive cells had appeared throughout the marrow cavity. We conclude that, at the proximal tibial metaphysis of female mice, estrogen-induced medullary sclerosis largely reflects a process of de novo medullary bone formation, possibly mediated by the generation of osteoblasts from bone marrow osteoprogenitor cells.