Molecular complexes, arguably the basic units carrying cellular function, can be visualized directly in their native environment by cryo-electron tomography. Here we describe a procedure for the detection of small, pleomorphic membrane-bound molecular complexes in cryo-tomograms by a hierarchical connectivity segmentation. Validation on phantom and real data showed above 90% true positive rates. This segmentation procedure is implemented in the Pyto software package, together with methods for quantitative characterization and classification of complexes detected by our segmentation procedure and for statistical analysis between experimental conditions. Therefore, the methods presented provide a means for the detection and quantitative interpretation of structures captured in cryo-electron tomograms, as well as for the elucidation of their cellular function. (C) 2016 Published by Elsevier Inc.