Microtubules (MTs) are hollow cylindrical polymers composed of alphabeta-tubulin heterodimers that align head-to-tail in the MT wall, forming linear protofilaments that interact laterally. We introduce a probe of the interprotofilament interactions within MTs and show that this technique gives insight into the mechanisms by which MT-associated proteins (MAPs) and taxol stabilize MTs. In addition, we present further measurements of the mechanical properties of MT walls, MT-MT interactions, and the entry of polymers into the MT lumen. These results are obtained from a synchrotron small angle x-ray diffraction (SAXRD) study of MTs under osmotic stress. Above a critical osmotic pressure, P(cr), we observe rectangular bundles of MTs whose cross sections have buckled to a noncircular shape; further increases in pressure continue to distort MTs elastically. The P(cr) of approximately 600 Pa provides, for the first time, a measure of the bending modulus of the interprotofilament bond within an MT. The presence of neuronal MAPs greatly increases P(cr), whereas surprisingly, the cancer chemotherapeutic drug taxol, which suppresses MT dynamics and inhibits MT depolymerization, does not affect the interprotofilament interactions. This SAXRD-osmotic stress technique, which has enabled measurements of the mechanical properties of MTs, should find broad application for studying interactions between MTs and of MTs with MAPs and MT-associated drugs.