The nupG gene, encoding one of the two active nucleoside-transport systems in Escherichia coli K-12, has been cloned on the multicopy plasmid pBR322 and derivatives thereof. The recombinant plasmids complemented a chromosomal nupG mutation. A genetic map was determined by digestion with restriction endonucleases and the nucleotide sequence of a 3-kb stretch of DNA has been determined on fragments cloned into M13 phages. An open reading frame of 1254 bp, encoding a protein with a calculated molecular mass of 45.333 kDa, was deduced to be the coding region of nupG. Minicell-forming strains carrying plasmids containing this gene were shown to produce a hydrophobic, membrane-bound polypeptide with an apparent molecular mass of approximately 43 kDa.