1. ATP-gated non-selective cation channels from the rat vas deferens (P2X1 receptors) were stably expressed in HEK 293 cells, assayed by patch clamp on the first day after passage of the culture, and found to have whole-cell current kinetics markedly faster in both activation and desensitization than those found in the native vas deferens tissue, in agreement with previous reports. 2. By the second day after passage of the culture, however, the whole-cell current kinetics of the expressed receptors shifted, slowing in both activation and desensitization. The kinetic change correlated with a change in phenotype of the host cells from round to flat, and the slower kinetics were similar to native P2X1 currents recorded from dissociated rat vas deferens smooth muscle cells. Two point mutations in a pore-like domain near or within the second transmembrane domain of the P2X1 receptor appeared to confer on the receptor the inability to effect this change in kinetics over time. 3. Treatment of cells on day 3 after passage with cytochalasins B or D caused a reversion to the rapid kinetics phenotype, implicating the actin cytoskeleton in the development of the native kinetics. P2X1 receptors may therefore require interaction with an intact actin cytoskeleton for native kinetics, and the mutants may be defective either in interaction with the actin skeleton or in coupling the interaction to gating.