SHG interferometry was used for the characterization of a rhodamine B derivative (RhB-1) in LB films. The RhB-1 LB films were deposited during the downstroke on fused silica substrates pre-coated with a stearic acid monolayer. It was found that the RhB-1 molecules deposited on the stearic acid monolayer were oriented with the hydrophilic head-group oriented toward the substrate. This orientation is the opposite of that expected for this deposition procedure, suggesting that the rhodamine B chromophore overturns during or after the deposition process.