To study the relationship of sequence to function for the phage lambda nutL transcriptional antiterminator, we cloned the 354-bp HincII lambda DNA fragment (coordinates 35261-35615; Daniels et al., in Lambda II, 1983, pp. 485-486 and 618-619) between the plac promoter and Rho-dependent or Rho-independent terminators (t) in the plac-t-galK plasmid derived from vector pKO3, and assayed the galK expression in Escherichia coli hosts in the presence or absence of the N gene product. The 354-bp fragment displayed the complete antitermination activity, as did several shorter fragments obtained by restriction cutting, exonucleolytic deletions and ligations. The minimal length of cloned and fully active nutL comprises 43 bp and consists (in the 5'-to-3' order) of a 10-bp sequence upstream from boxA, the 8-bp boxA (5'-CGCTCTTA-3'), the 7-bp A-B spacer between boxA and boxB; the 17-bp boxB (nutL core; 5'-AGCCCTGAAGAAGGGCA-3') and 1-bp downstream from the nutL core. Deletion of the 10-bp sequence upstream from boxA reduces anti-termination by 40%. Deletion of both that sequence and boxA reduces antitermination by 90% at both 30 degrees C and 42 degrees C. Most of the deletions entering boxB abolish antitermination. Also, some of the small internal deletions within the 7-bp A-B spacer region have a strong negative effect on the nutL function, when transcription is from the plac promoter.