Laser Doppler flowmetry (LDF) has been successfully used in clinical and experimental settings to evaluate bone perfusion but unanswered questions regarding its capabilities and limitations still remain. This study was undertaken to determine absorption of He-Ne laser light (632.8 nm) and maximum depth for flow assessment (threshold thickness) under optimal conditions in bone. Light transmittance in bovine bone samples of femora and tibia was measured after each step of grinding and depth of penetration calculated. The threshold thickness was obtained by placing the same samples in a flow chamber where a solution of 2% latex circulated beneath; flow was detected by a laser Doppler probe resting on top of the sample. The results showed a significantly higher depth of penetration for trabecular than for cortical bone. A regression analysis showed a high correlation between the inorganic fraction of the bone and the depth of penetration. The maximum depth at which the laser Doppler probe can evaluate flow in bone conditions was found to be 2.9 +/- 0.2 mm in cortical bone, 3.5 +/- 0.3 mm in bone covered by 1 mm cartilage and 3.5 +/- 0.2 mm in trabecular bone. The study showed the limitations of LDF in bone and their correlations to various bone properties.