As large genomic and proteomic datasets are generated from homogenates of various tissues, the need for information on the spatial localization of their encoded products has become more pressing. Matrix-assisted laser desorption-ionization (MALDI) imaging mass spectrometry (IMS) offers investigators the means with which to unambiguously study peptides and proteins with molecular specificity, and to determine their distribution in two and three dimensions. In the past few years, several parameters have been optimized for IMS, including sample preparation, matrix application and instrumental acquisition parameters (Box 1). These developments have resulted in a high degree of reproducibility in mass accuracy and peak intensities (Supplementary Fig. 1 online). Recently, we have optimized our protocol to be able to increase the number of molecular species analyzed by collecting two sets of sections, covering one set of sections with sinapinic acid for optimal detection of proteins and adjacent sections with 2,5-dihydroxybenzoic acid (DHB) matrix for the optimal detection of low-mass species, including peptides. Approximately 1,000 peaks can be observed in each dataset (Fig. 1). Furthermore, the sections are collected at an equal distance, 200 mum instead of 400-500 mum used previously, thus enabling the use of virtual z-stacks and three-dimensional (3D) volume renderings to investigate differential localization patterns in much smaller brain structures such as the substantia nigra and the interpeduncular nucleus. Here we present our optimized step-by-step procedure based on previous work in our laboratory, describing how to make 3D volume reconstructions of MALDI IMS data, as applied to the rat brain.