The lamellar bone's strength is mainly affected by the organization of its mineralized collagen fibers and material composition. In the present study, Raman microspectroscopic and imaging analyses were employed to study a normal human femoral midshaft bone cube-like specimen with a spatial resolution of ∼ 1-2 μm. Identical bone lamellae in both longitudinal and transverse directions were analyzed, which allowed us to separate out orientation and composition dependent Raman lines, depending on the polarization directions. This approach gives information about lamellar bone orientation and variation in bone composition. It is shown that the ν1 PO4 to amide I ratio mainly displays lamellar bone orientation; and ν2 PO4 to amide III and CO3 to ν2 PO4 ratios display variation in bone composition. The ν2 PO4 to amide III ratio is higher in the interstitial bone region, whereas the CO3 to ν2 PO4 ratio has lower values in the same region. The present study provides fresh insights into the organization of a lamellar bone tissue from two orthogonal orientations. © 2007 Elsevier Inc. All rights reserved.