Atomic force microscopy was used to investigate the surface morphology and transverse stiffness of myofibrils from Drosophila indirect flight muscle exposed to different physiologic solutions. I- and A-bands were clearly observed, and thick filaments were resolved along the periphery of the myofibril. Interfilament spacings correlated well with estimates from previous x-ray diffraction studies. Transverse stiffness was measured by using a blunt tip to indent a small section of the myofibrillar surface in the region of myofilament overlap. At 10 nm indention, the effective transverse stiffness (K( perpendicular)) of myofibrils in rigor solution (ATP-free, pCa 4.5) was 10.3 +/- 5.0 pN nm(-1) (mean +/- SEM, n = 8); in activating solution (pCa 4.5), 5.9 +/- 3.1 pN nm(-1); and in relaxing solution (pCa 8), 4.4 +/- 2.0 pN nm(-1). The apparent transverse Young's modulus (E( perpendicular)) was 94 +/- 41 kPa in the rigor state and 40 +/- 17 kPa in the relaxed state. The value of E( perpendicular) for calcium-activated myofibrils (55 +/- 29 kPa) was approximately a tenth that of Young's modulus in the longitudinal direction, a difference that at least partly reflects the transverse flexibility of the myosin molecule.