Atomic force microscopy (AFM) was used to study the surface topology and elasticity distribution of vertebrate myofibrils in physiological salt buffer. Images were recorded from isolated myofibrils of rabbit skeletal muscle. Z-disc, M-bands, A-bands, and I-bands were clearly observed and regularly spaced structures likely representing myosin filaments were readily detectable on the myofibrillar surface. A histogram analysis of the average lateral interfilament spacing detected perpendicular to the myofibril axis showed a d_1,0 (distance between the lattice planes made by myosin filaments) of 40.92 ± 4.45 nm (mean ± SD), identical to the d_1,0 of 40.3 nm (SL = 2.2 mm) for relaxed rabbit psoas muscle known from x-ray diffraction. Thus, AFM can provide nm-resolution topographical information on the myofibrillar surface. Force mapping revealed periodic transverse stiffness patterns along psoas myofibrils, which were more distinctive in rigor compared to relaxed state. A-band transverse stiffness at 25 nm indentation was higher in rigor (4.2 ± pN/nm) than in relaxed myofibrils (0.4 ± pN/nm). Titin digestion by low-dose trypsin decreased the apparent elasticity modulus in the A-bands of rigor myofibrils from 135.36 kPa to 101.56 kPa (at 20 nm indentation), but did not effect this parameter in relaxed myofibrils. Comparing relaxed psoas and diaphragm myofibrils showed no differences regarding the apparent elasticity modulus. This result questions the proposed role of titin as a generator of lateral force component