Ca²⁺ signals from activated Ca²⁺ channels (CatSper) and mobilisation of Ca²⁺ stores regulate human sperm cell behaviour as they ascend the female tract. I investigated the effects on human sperm [Ca²⁺]i and behaviour of CatSper channel activation (alkaline pH and progesterone) and Ca²⁺-store mobilisation (4-aminopyridine, thimerosal) using a fluorescence plate reader and CASA. Extracellular alkalinisation raised pH¬i (pHi = 6.9 and 7.2 at pHo7.4 and 8.5 respectively), caused tonic elevation of [Ca²⁺]i, which was partially inhibited by CatSper block and increased the proportion of hyperactivated cells (from 1.8±0.5 to 10.5±1.6%; n=34, P=1x10⁻⁷). Progesterone elevated [Ca²⁺]i but caused negligible hyperactivation. Co-application of these stimuli revealed little, if any, synergistic interaction. Ca²⁺-store mobilisation (4-aminopyridine) caused prolonged [Ca²⁺]i elevation and was associated with strong hyperactivation. Analysis of [Ca²⁺]i and hyperactivation data from 24 different conditions in this study showed a continuous relationship between [Ca²⁺]i and hyperactivation. The strong hyperactivating effect of store mobilisation (compared to CatSper activation) may reflect opening of store-operated channels. Human sperm behaviour assessed over a 180 s recording revealed regular ‘switching’ between progressive and various hyperactivated types. Mobilisation of Ca²⁺ stores potently increased hyperactivated behaviour and suppressed the rate of behavioural switching.