Although interstitial fluid flow has been suggested to play a role in bone adaptation and metabolism, the constituents and ultrastructure of this interstitial fluid pathway are not well understood. Bone's lacunar–canalicular porosity is generally believed to be a continuous interstitial fluid pathway through which osteocytes sense external mechanical loading as well as obtain nutrients and dispose of wastes. Recent electron microscopy studies have suggested that a fiber matrix surrounds the osteocytic cell processes and fills this pericellular fluid space. However, studies injecting tracer molecules into the bone vasculature have provided conflicting results about the pore size or the fiber spacing of the interstitial fluid pathway. In addition, whether the smaller collagen–apatite porosity in adult bone is also a continuous fluid pathway is still unclear. To delineate bone's interstitial fluid pathway, four tracers of various size were injected into rats:​ reactive red (approximately 1 nm), microperoxidase (MP, approximately 2 nm), horseradish peroxidase (HRP, approximately 6 nm), and ferritin (approximately 10 nm). Five minutes after injection, the tibiae were harvested and processed using histological protocols optimized to minimize processing time to reduce possible redistribution of tracer molecules. The number of blood vessels and osteocytic lacunae labeled with the tracers per unit bone area was then measured for mid-diaphysial cross-sections of the tibia. While none of the tracers was detected within the mineralized bone matrix (the collagen–apatite porosity) using light microscopy, all the tracers except ferritin were found to pass through the canaliculi and appear in the osteocytic lacunae. These results indicate that while small tracers (< 6 nm) readily pass through the lacunar–canalicular porosity in the absence of mechanical loading, there appears to be an upper limit or cutoff size between 6 and 10 nm for molecular movement from bone capillaries to osteocytic lacunae in rat long bone. This range of pore size contains the most likely fiber spacing (approximately 7 nm) that has been proposed for the lacunar–canalicular annular space based on the presence of a proteoglycan fiber matrix surrounding the osteocyte.