Calcium (Ca(2+)) is an absolute requirement for a decisive sperm function event: the acrosome reaction (AR). Physiologically, sperm capacitation is a prerequisite for this specialized exocytosis and both events are intimately related. In an effort to separate capacitation from AR, we have been using a modified sperm incubation medium where Ca(2+) is replaced by Strontium (Sr(2+)). The aim of this report is to analyze with more detail the difference between sperm incubated with Ca(2+) or Sr(2+) in several events. We found that sperm undergo the capacitation-related changes in the chlortetracycline (CTC) pattern and tyrosine phosphorylation, and also bind to the zona pellucida (ZP) when using Sr(2+)-instead of Ca(2+)-containing media. However, the spontaneous AR typical of hamster sperm does not take place in Sr(2+)-medium, even if sperm are previously capacitated with Ca(2+). Nevertheless, Sr(2+) was able to sustain AR when cells were treated with thapsigargin or depolarized with K(+) in Na(+)-depleted medium. Considering that the absence of Na(+) increased spontaneous AR in Sr(2+)-medium, we tested whether Na(+)-transport systems could be involved in the inability of Sr(2+)-incubated sperm to undergo AR. We found that when sperm incubated in Sr(2+)-medium are treated with amiloride to inhibit epithelial Na(+) channel (ENaC), they are able to undergo spontaneous AR. The same result was obtained when analyzing AR on the ZP. On the contrary, addition of ouabain (a Na(+)/K(+)-ATPase inhibitor) or DIDS (a Na(+)/HCO3(-) co-transporter inhibitor) showed no effect. These results suggest that, differing from what happens in Ca(2+)-incubated sperm, cells incubated in Sr(2+)-modified medium would have an active ENaC.