A physical map of the chromosome of the erythromycin-producing actinomycete Saccharopolyspora erythraea NRRL 2338 has been constructed using the restriction enzymes AseI and DraI. The map was constructed by a variety of methods including linking clone analysis, cross-hybridizations using labelled macrorestriction fragments, gene probing, two-dimensional PFGE and restriction enzyme site generation. Analysis of the individual macrorestriction patterns of the 17 AseI-, 6 DraI- and 22 AseI/DraI-digested fragments indicated a chromosome size of about 8 Mb. Linking clones for five contiguous AseI fragments were obtained, covering 32% of the chromosome. The linkage of an additional eight AseI fragments was aided by the finding that the rRNA operons of S. erythraea contain an AseI site within the 16S (rrs) gene. Generation of S. erythraea strains that contain additional DraI sites within selected AseI fragments, followed by PFGE analysis and Southern hybridization to determine specific linkages, facilitated the completion of the AseI map. The entire DraI map was constructed by gene probing and cross-hybridizations. PFGE analysis of agarose-embedded DNA prepared in either the presence or absence of proteinase K suggested that the S. erythraea NRRL 2338 chromosome is linear. A total of 15 genes or gene clusters were mapped to specific AseI and DraI fragments, including the erythromycin-biosynthetic gene cluster and the rRNA operons.