Diverse polyamine transport systems have been described in different cells, but the molecular entities that mediate polyamine influx and efflux remain incompletely defined. We have previously demonstrated that spermidine efflux from oocytes is a simple electrodiffusive process, inhibitable by external Ca2+, consistent with permeation through a membrane cation channel. Hemi-gap junctional channels in Xenopus oocytes are formed from connexin 38 (Cx38), and produce a calcium-sensitive (Ic) current that is inhibited by external Ca2+. Spermidine efflux is also calcium sensitive, and removal of external calcium increases both Ic currents and spermidine efflux in Xenopus oocytes. Injection of Cx38 cRNA or Cx38 antisense oligonucleotides (to increase or decrease, respectively, Cx38 expression) also increases or decreases spermidine efflux in parallel. Spermidine efflux has a large voltage-dependent component, which is abolished with injection of Cx38 antisense oligonucleotides. In addition, spermidine uptake is significantly increased in Cx38 cRNA-injected oocytes in the absence of external calcium. The data indicate that hemi-gap junctional channels provide the Ca2+-inhibited pathway for electrodiffusive efflux of polyamines from oocytes, and it is likely that hemi-gap junctional channels provide Ca2+ and metabolism-sensitive polyamine permeation pathways in other cells.