The association of the bacteriophage T4-encoded AsiA protein with the final sigma(70) subunit of the Escherichia coli RNA polymerase is one of the principal events governing transcription of the T4 genome. Analytical ultracentrifugation and NMR studies indicate that free AsiA is a symmetric dimer and the dimers can exchange subunits. Using NMR, the mutual recognition sites on AsiA and final sigma(70) have been elucidated. Residues throughout the N-terminal half of AsiA are involved either directly or indirectly in binding to final sigma(70) whereas the two highly conserved C-terminal regions of final sigma(70), denoted 4.1 and 4.2, constitute the entire AsiA binding domain. Peptides corresponding to these regions bind tightly to AsiA individually and simultaneously. Simultaneous binding promotes structural changes in AsiA that mimic interaction with the complete AsiA binding determinant of final sigma(70). Moreover, the results suggest that a significant rearrangement of the dimer accompanies peptide binding. Thus, both conserved regions 4.1 and 4.2 are intimately involved in recognition of AsiA by final sigma(70). The interaction of AsiA with 4.1 provides a potential explanation of the differential abilities of DNA and AsiA to bind to free final sigma(70) and a mechanistic alternative to models of AsiA function that rely on binding to a single site on final sigma(70).