In ICSI procedures, it is well known that the selection of viable (live) spermatozoa and certain types of immobilization prior to injection is very important for obtaining successful results, but unfortunately there are rare situations when only immotile spermatozoa are available (such as in severe asthenozoospermia or necrozoospermia). In such cases, failure of oocyte activation after ICSI often occurs and may be due to the lack of SOAF (sperm-borne oocyte activating factor) activity. In order to investigate the SOAF activities of dead spermatozoa, mouse and human spermatozoa were immobilized (killed by sonication), maintained in THF medium for varying time intervals (up to 72 h) and then injected into mature unfertilized mouse oocytes. Injected mouse oocytes were examined for their activation, development into blastocysts and Ca2+ responses by imaging and confocal laser scanning microscope. The rates of oocyte activation, blastocyst development and normal patterns of Ca2+ oscillation from the killed-sperm-injected oocytes decreased gradually in accordance with the maintenance interval between sonication and injection. For injection with mouse sonicated spermatozoa, the rate of normal Ca2+ oscillations declined first (after a 3 h maintenance interval) and then blastocyst development was gradually obstructed (after approx. 10 h). The oocyte activation-inducing ability of dead spermatozoa was maintained for a relatively long period, but began to decline after 20 h. The activation rates and Ca2+ response of the oocytes that were injected with human sonicated spermatozoa decreased earlier than those injected with mouse spermatozoa. Although the oocyte activation-inducing ability was maintained for a relatively long time after the death of the spermatozoa, embryo development into blastocysts and the rate of normal Ca2+ oscillations declined after a short maintenance interval between sonication and injection. The Ca2+ response seemed to be the most sensitive indicator for the evaluating the SOAF activity of dead (killed) spermatozoa.