While matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has revolutionized the manner by which many large molecules are characterized, the highly variable appearance of MALDI mass spectra remains a concern. We have developed MALDI-based imaging as a diagnostic tool for examining the relationships between preparation strategy, sample morphology, and spectral quality. The imaging protocol involves the automated acquisition of mass spectra at 400-1600 positions within a single sample, followed by off-line processing and image display. Several sample types have been characterized, including a simple peptide mixture prepared in dried droplets of 2,5-dihydroxybenzoic acid and in thin films of alpha-cyano-4-hydroxycinnamic acid as well as a complex biological sample consisting of intact peptidergic neurons from the marine mollusk Aplysia californica. Imaging experiments provide a wealth of unbiased information concerning sample defects, spectral reproducibility, mass accuracy, differential analyte distributions, and the validity of internal standards.