Novel biocompatible macromolecular vectors were developed that not only enable transport of bioactive cargo across the cell membrane but also control the delivery into defined intracellular compartments. This work describes the synthesis and design of two non-peptidic fluorescently labeled Newkome-type dendrimers, differentiated over a varied alkyl spacer with guanidine end moieties. The internalization of the fluorescein-labeled molecular transporter into mammalian cells showed strong subcellular localizations, evident with both live cells and fixed cells costained with DAPI, a nuclear stain. We observed that the subcellular distribution of these vectors varied significantly, as one of the vectors concentrates in the nucleus (FD-1) while the other (FD-2) concentrates in the cytosol. All experiments performed with NIH-3T3 fibroblasts and human microvascular endothelial cells (HMEC) showed similar results. The differential localization patterns of the two molecular transporters can be controlled through the variation of alkyl spacer length at the terminal generation of the dendrimer. Intracellular delivery of bioactive entities into specific subcellular locations, utilizing this practical approach, might overcome limitations in drug delivery and pioneer future technologies in drug transport.