In the development of oligoarginine-based prodrugs with self-cleavable spacers for intestinal absorption, we previously reported a series of spacers with variable half-lives of parent compound release based on a neighboring group participation mechanism from an amino acid side-chain structure next to the succinyl moiety. In the present study, to diversify the half-life of the spacer, we first synthesized several additional fluorescein isothiocyanate ethanolamine (FE)-heptaarginine conjugates (4d--g) and evaluated their conversion time. To investigate the overall cellular uptake of FE-heptaarginine conjugates, the cellular uptakes of FE-heptaarginines 4a and 4b possessing the longest and shortest half-lives, respectively, were evaluated using HeLa cells by confocal microscopy and flow cytometry. Conjugate 4a with a longer half-life was more efficiently taken up by the cells than conjugate 4b. However, in term of the transport rate of parent FE 1 in in vitro Caco-2 cell permeation assay, conjugate 4b with a short half-life could function more efficiently that conjugate 4a. To understand the reason for this discrepant finding, fluorescence on the basal side medium after treatment with conjugate 4b in the permeation assay was determined. It became apparent that the fluorescence was mostly from the parent FE 1 itself, and not conjugate 4b, suggesting that the conjugate was cleaved inside the cells. Moreover, the conversion time of conjugate 4b (t1/2=9.4 min at pH 7.4) was significantly extended in slightly acidic media. These results suggest that the conversion rate was slowed in the relatively acidic endosomal environment where the conjugate was transferred after endocytosis, and resulted in a favorable migration time across the cells. The other conjugates, including conjugate 4a, were more stable inside of the cell, resulting in very long conversion times that were ineffective in increasing the permeation rate. Therefore, spacers with shorter half lives, in order to produce a larger amount of the parent compound inside the cells are promising development for effective oligoarginine-based cargo-transporter systems to enhance intestinal absorption of parent drugs with low permeability.