PurposeThis study investigated the effects of esterification and increased lipophilicity on cellular penetration, accumulation and retention in ARPE-19-nic cells using ester functionalized rhodamine B dyes.MethodsRhodamine B was esterified to generate four dyes with increasing lipophilicity. Cellular uptake, retention and mitochondrial localization were investigated in vitro using ARPE-19-nic cells using direct intracellular and extracellular and mitochondrial fluorescence quantitation, confocal and high-resolution live cell imaging and co-localization with Mito-GFP.ResultsCellular penetrance, mitochondrial accumulation, and retention of the esterified dyes were increased in ARPE-19-nic cells compared with the nonesterified parent dye by direct fluorescence quantitation. Imaging demonstrated intracellular accumulation was confined to mitochondria as confirmed by colocalization with Mito-GFP.ConclusionsEsterification is an effective way to increase lipophilicity of a dye to improve cellular penetration of chemical entities. These observations may be key to improving retinal drug delivery for retinal pigment epithelium–based diseases.Translational RelevanceUnderstanding the intracellular distribution of drugs into retinal pigment epithelium cells is a critical component for identifying potential therapies for retinal pigment epithelium–based diseases.