Planaria flatworms regenerate new heads and tails after amputation by differentiation of stem cells (neoblasts), but the cues that drive spatially appropriate differentiation (head versus tail) are not clear. Previous work identified a voltage gradient inside planaria, with the tail mesenchyme tissue positive relative to the head (Annand, 2014). A membrane potential gradient also exists in planarian epithelial cells, which is relatively more depolarised at the head (Beane et al., 2011). We therefore tested the hypothesis that a bioelectrical gradient and voltage-gated chloride ion transport contribute to axial regeneration polarity. Dugesia japonica (4-6 mm) were amputated on ice into equal head, trunk and tail fragments. Trunk fragments were immobilised in 7% methylcellulose and exposed to a DC electric field of 150 mV/mm for 1h with the polarity either parallel or antiparallel to the fragment’s endogenous voltage gradient. After treatment fragments were transferred to planaria water for 2 weeks to regenerate. Appearance of eyespots was used to score head regeneration in trunks of 5 worms in 3 independent experiments. Frequencies were compared with a D test (Bailey, 1981); significance at P<0.05. Eye regeneration was first apparent on day 4 (20% had eyes) in control trunks and on day 5 (50% with eyes) when the EF was parallel to the endogenous internal EF (anterior facing the cathode) but it was delayed until day 7 (51% had eyes) in trunks exposed to an antiparallel external EF. 100% of regenerating fragments had eyes by day 6 for control and parallel EF conditions but eye appearance was delayed until day 8 for the antiparallel EF group. Fragments survived better (93%) in control and parallel EF groups than the antiparallel EF group (60%; P<0.001), in which ectopic eyes were observed. The role of voltage-gated chloride transport was tested by incubating trunk and tail fragments in planaria water with 10 µM 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) or 0.1% DMSO (vehicle) throughout the whole 2 week regeneration period. Trunk and tail fragments were pooled from 4 control experiments and 5 DIDS and DMSO experiments run in parallel; 5 worms each. Head regeneration was delayed by DIDS; 7 days after amputation 100% of controls and 89% of DMSO controls (not statistically significant) had eyes, but only 58% (P<0.001 compared to DMSO) of the DIDS group had eyes. By day 10, 100% of the control and DMSO groups had eyes and 90% of the DIDS group had eyes and some of those were ectopic. We conclude that the inherent bioelectrical gradient and epithelial chloride transport each contribute to axial regeneration polarity in planaria. The ability to drive neoblasts selectively down distinct lineages may have clinical relevance for mammalian regeneration, suggesting strategies that target ionic flux and electrical properties specific to regenerating tissues.