The exposure of freshly spawned, immotile carp sperm to hypoosmotic media triggers the initiation of calcium-dependent flagellar motility. Intracellular calcium concentration has been thought to be the critical component in motility initiation, possibly acting through a novel signalling pathway. The sensitivity of sperm cells to changes of osmolality of the environment raises the question whether a mechanoregulated osmosensitive calcium pathway is involved in the activation mechanism of carp sperm motility. The sperm cells are in a depolarized state in the seminal plasma (Ψ = –2.6 ± 3 mV) and they hyperpolarize upon hypoosmosis-induced activation of motility (Ψ = –29 ± 4 mV). The intracellular sodium [Na+]i, potassium [K+]i and calcium [Ca2+]i ion concentrations were determined in quiescent cells, and at 20, 60 and 300 s after activation. The [Na+]i and [K+]i of the quiescent cells were similar to the [Na+]e and [K+]e of the seminal plasma. Following hypoosmotic shock-induced motility, both [Na+]i and [K+]i decreased to one-fourth of the initial concentration. The [Ca2+]i doubled at initiation of the motility of the sperm cells and remained unchanged for 5 min. Bepridil (50–250 μM), a blocker of the Na+/Ca2+ exchanger, blocked carp sperm motility reversibly. Gadolinium, a blocker of stretch-activated channels (10–20 μM), inhibited sperm motility in a dose-dependent manner and its effect was reversible. Hypoosmotic shock fluidized the membrane and gadolinium treatment made it more rigid in both quiescent cells and hypotonic shock treated but immotile sperm cells. Based on these observations, it is suggested that, besides the well-known function of potassium and calcium channels, stretch-induced conformational changes of membrane proteins are also involved in the sperm activation mechanism of common carp.