To determine whether overlapping Fis sites can be simultaneously bound by Fis, we synthesized self-complementary DNA oligos with strong Fis sites that overlap by either 7 or 11 base pairs (Fig. 4a,b) or that were separated by 23 base pairs (Fig. 4c) and tested their properties by gel shift. Neither the 11 nor the 7 overlapping sites showed a doubly-shifted band, even at an extremely high Fis/DNA ratio and with exceptionally strong (>12 bits) Fis binding sites (Fig. 5), suggesting that only one Fis molecule could bind to each DNA fragment. The DNA fragment with two Fis sites separated by 23 base pairs did double shift (Fig. 5), demonstrating that two well separated Fis sites can simultaneously bind two Fis molecules. However, Fis can create a ladder of complexes on non-specific sequences [50], and this might account for the double-shifts. Under our conditions with short DNAs, a non-specific (all positions < 1 bit) 66 bp DNA fragment barely shifted at high Fis concentration (data not shown) so the secondary shifts were not from non-specific binding. These results demonstrate that Fis sites separated by 7 or 11 bases cannot be bound simultaneously.