The pollen tube has been widely used to study the mechanisms underlying polarized tip growth in plants. A steep tip-to-base gradient of free cytosolic calcium ([Ca2+]cyt) is essential for pollen-tube growth. Local Ca2+ influx mediated by Ca2+-permeable channels plays a key role in maintaining this [Ca2+]cyt gradient. Here, we developed a protocol for successful isolation of spheroplasts from pollen tubes of Pyrus pyrifolia and identified a hyperpolarization-activated cation channel using the patch-clamp technique. We showed that the cation channel conductance displayed a strong selectivity for divalent cations, with a relative permeability sequence of barium (Ba2+)≈Ca2+>magnesium (Mg2+)> strontium (Sr2+)>manganese (Mn2+). This channel conductance was selective for Ca2+ over chlorine (Cl-) (relative permeability PCa/PCl=14 in 10 mM extracellular Ca2+). We also showed that the channel was inhibited by the Ca2+ channel blockers lanthanum (La3+) and gadolinium (Gd3+). Furthermore, channel activity depended on extracellular pH and pollen viability. We propose that the Ca2+-permeable channel is likely to play a role in mediating Ca2+ influx into the growing pollen tubes to maintain the [Ca2+]cyt gradient.