The Stokes shift of tryptophan (Trp) fluorescence from layers of the lipid-containing bacteriophage ϕ6 are compared to determine the relative effect of the layers on virus hydrophobicity. In the inner most layer, the empty procapsid (PC) which contains 80% – 90% of the virion Trp residues, λmax = 339.8 nm. The PC emission is substantially more red-shifted than the other ϕ6 layers and nearer to that of the Pseudomonad host cell than the other ϕ6 layers. The Trp emission from the nucleocapsid (NC) with λmax = 337.4 nm, is blue shifted by 2.4 nm relative to the PC although the number of Trp in the NC is identical to the PC. This shift represents an increase in Trp hydrophobicity, likely a requirement for the maintenance of A-form dsRNA. Fluorescence from the completely assembled virion indicates it is in a considerably more hydrophobic environment with λmax = 330.9 nm. Density measurements show that the water content in the NC does not changed during envelope assembly, therefore the blue-shifted ϕ6 emission suggests that the envelope changes the PC environment, probably via the P8 layer. This change in hydrophobicity likely arises from charge redistribution or envelope-induced structural changes in the PC proteins.