Hoechst 33342 and Ethidium Homodimer should colocalize perfectly and in the same plane as one another, even though one is a minor groove binding dye and the other is an intercalating dye.

My best guess is that either your microscope objectives are not chromatic aberration-corrected or, possibly (but I know you say they are not), that your filters are not aligned.

I've used the dyes extensively together and in my experience on a laser scanning confocal microscope with chromatically corrected PlanFluor objectives they localize entirely.

Thanks for your answer. We still think everything with the scope is fine, but one idea we have had is that some cell types may not be permeable to the Hoechst dye. It seems that the Ethidium Homodimer is actually labeling a distinct population of cells, which I saw when both Hoechst labeled and Ethidium Homodimer labeled nuclei were also marked by an RNA dye. So as a follow up, do you know of any circumstances in which Hoechst 33342 can be cell-impermeable? 

Hoechst 33342 should be permeable to all mammalian cell types to my knowledge. Is it possible that your Hoechst has gone bad? Have you imaged your samples on a different microscope?

Are you certain you have H33342 and not H33258? H33258 is much less permeable due to differences in hydrophobicity.