The prototype strain of the human adenovirus type 35 (AV35) was examined. BamHI, EcoRI, HindIII, KpnI, PstI, and SalI restriction endonucleases were used for the mapping of DNA fragments. Three original maps were constructed, and previously published maps were somewhat modified. A PstI-specific fragment library was also prepared and characterized using the pBR322/E. coli system. Some of the recombinants seem to be applicable for rapid DNA diagnostics, and for the comparative mapping of type- and subgroup-specific DNA sequences. The comparative presentation of physical maps of subgroup B human adenoviruses might improve the efficiency of genotyping of adenoviruses using restriction endonucleases.