We have used atomic force microscopy (AFM) to investigate the local mechanical and structural properties of microtubules under physiologically relevant conditions. AFM provides a unique tool for these types of studies because of its ability to probe the microtubule locally, and hence the ability to resolve variations in structure and mechanical properties within a single microtubule. We have imaged stabilized microtubules bound to 3-aminopropyltriethoxylsilane-coated glass and mica surfaces. We have developed protocols for imaging microtubules with little visible contamination. Comparing the heights from AFM data from microtubules imaged dry to those imaged under buffer, we can differentiate between structurally intact microtubules and collapsed microtubules. We have also intentionally collapsed microtubules by applying force with the AFM probe tip in order to estimate their mechanical stability. We find that microtubules polymerized under different conditions can vary markedly in their stiffness, and that the stiffness varies along the length of the microtubule.