Here we describe software tools for the automated detection of DNA restriction fragments resolved on agarose fingerprinting gels. We present a mathematical model for the location and shape of the restriction fragments as a function of fragment size, with model parameters determined empirically from "marker" lanes containing molecular size standards. Automated identification of restriction fragments involves several steps, including: image preprocessing, to put the data in a form consistent with a linear model; marker lane analysis, for determination of the model parameters; and data lane analysis, a procedure for detecting restriction fragment multiplets while simultaneously determining the amplitude curve that describes restriction fragment amplitude as a function of mobility. In validation experiments conducted on fingerprinted and sequenced Bacterial Artificial Chromosome (BAC) clones, sensitivity and specificity of restriction fragment identification exceeded 96% on restriction fragments ranging in size from 600 base pairs (bp) to 30,000 bp. The integrated suite of software tools, written in MATLAB and collectively called BandLeader, is in use at the BC Cancer Agency Genome Sciences Centre (GSC) and the Washington University Genome Sequencing Center, and has been provided to the Wellcome Trust Sanger Institute and the Whitehead Institute. Employed in a production mode at the GSC, BandLeader has been used to perform automated restriction fragment identification for more than 850,000 BAC clones for mouse, rat, bovine, and poplar fingerprint mapping projects.