Frameshift mutations were induced by proflavin in the rIIB gene of bacteriophage T4. rIIB DNA from each of 48 independent frameshifts was inserted into M13mp8 and sequenced. Two-thirds of the frameshifts (33/48) lie contiguous to one another in 10 base pairs of the rIIB sequence. This hotspot differs markedly from previously characterized mutagen-induced frameshift hotspots. Distinctive features of the hotspot include the absence of locally repetitive sequences, particularly G X C runs, and the fact that many different sequence changes are induced within the hotspot sequence at appreciable frequencies. Among the 33 mutants at the hotspot, 8 distinguishable DNA sequence changes were seen. All of the mutations were deletions of a single base or duplications of one or more bases. Duplications were more frequent than deletions. The patterns of the base sequence changes suggest that two specific phosphodiester bonds within the hotspot sequence are sites at which proflavin-induced mutation is initiated.