The inner ear of the embryonic chick forms an oval-shaped sac or otocyst, on Embryonic Day 3, which contains presumptive sensory and support cells. After 3 weeks in organ culture the otocyst had sensory epithelia with an average of 325 ± 41 hair cells. Using light and transmission electron microscopy most of these cells were identified morphologically as type II vestibular hair cells. Whole-cell tight-seal recordings, using potassium chloride-filled micropipetes, showed that mature cultured hair cells had four different types of K currents. These included: a voltage-gated delayed rectifier K + current ( I K), an inactivating K + current ( I A), a calcium-dependent K + current ( I K(Ca)), and a K + inward rectifier ( I IR). These currents were similar to those recorded from cristae ampullares cells isolated from 2- to 3-week-old posthatched chicks. We also determined the timing of K + current acquisition in vitro. Initially, recordings showed that cells isolated from Embryonic Day 3 otocysts had no voltage-dependent outward currents at physiological membrane potentials. Eventually, K + currents were acquired in the order of: I K and I IR after 9 days, I A after 12 days, and I K(Ca) after 17 days in vitro . In addition, recordings using cesium chloride-filled micropipetes showed that there were two types of inward currents that were elicited in response to membrane depolarizations. These two currents included a rapidly activating, noninactivating Ca 2+ current and a tetredotoxin-sensitive Na + current. Both currents were elicited in hair cells grown in vitro for 13 days. Although I Na was previously unreported in avians, both I Na and I Ca were also represented in hair cells isolated from the cristae ampullares of the posthatched chick. These results indicate that hair cells can acquire voltage-gated currents in vitro which are qualitatively similar to ionic currents found in crista ampullaris cells that differentiate in vivo. Thus, this organ culture system provides a means to study regulation of ionic currents in developing hair cells.