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Four lambda clones (λG11, λC4, λG14 and λ G17) from the porcine MHC class III region were labelled with either biotin- 14-dATP or digoxigenin-11-dUTP and hybridized in two different combinations to DNA fibres. The latter were prepared from agarose-embedded porcine peripheral lymphocytes lysed with proteinase K, then spread and fixed on poly-L-lysine-coated slides. Hybridization signals thus obtained confirm the order of the clones previously reported using pulsed-field gel electrophoresis (PFGE). Measurements of probe sizes, gap distances between probes and total length of DNA encompassing the probes were made. Three different methods, namely relative length, Watson–Crick standard and probe size standard-based conversions, were used to estimate the parameters in kilobases. These methods of data conversion were compared with each other and with the available PFGE data, and their utility and accuracy were evaluated.