The DNA sequence of the F plasmid origin of conjugal DNA transfer, oriT , has been determined. The origin lies in an intercistronic region which contains several inverted repeat sequences and a long AT-rich tract. Introduction of a nick into one of the DNA strands in the oriT region precedes the initiation of conjugal DNA replication, and the position of the strand-specific nicks acquired by a lambda oriT genome upon propagation in Flac-carrying cells has been determined. The nicks were not uniquely positioned, rather there was a cluster of three major and up to 20 minor sites: the biological significance of this observation is not yet fully clear. Nine independent point mutations which inactivate oriT function have been sequenced and found to alter one or other of two nucleotide positions which lie 14 and 19 bp to one side of the rightmost (as drawn) major nick site. These key nucleotides may lie in a recognition sequence for the oriT endonuclease, since mutations at these sites prevent nicking at oriT .