Aim: The increase in the concentration of cytosolic-free calcium ([Ca2+]i) induced by follicular fluid or progesterone has been reported to promote an acrosome reaction and alternation in several motion parameters in human sperm (hyperactivation). We previously reported that populations of sperm in cell suspension obtained from infertile men with abnormal morphology exhibited lower mean peak progesterone-evoked [Ca2+]i compared with morphologically normal sperm using cell-suspension methods. In the present study, the change in [Ca2+]i in individual normally and abnormally shaped spermatozoa was compared. Methods: The change in [Ca2+]i induced by human follicular fluid in individual spermatozoa with normal and abnormal morphology was compared using the fluorescent calcium-sensitive dye fluo-3/AM. The spatial distribution of the increase in [Ca2+]i in single sperm was also investigated. Results: The [Ca2+]i of normally shaped spermatozoa increased rapidly after the administration of human follicular fluid. The response reached a peak within 2-3s and then slowly declined to a plateau phase. The baseline and peak fluorescence in spermatozoa with abnormal morphology was lower when compared with normal spermatozoa. The follicular-fluid-induced increase in [Ca2+]i (expressed as a percentage increase in [Ca2+]i over basal) in morphologically abnormal sperm was 39.2±5.3% (n=107, mean±standard error), which was smaller than that of morphologically normal sperm (61.6±5.7%, n=100, P<0.005) from seven healthy donors. The follicular-fluid-induced [Ca2+]i increases observed in sperm with morphologically abnormal mid-pieces (20.9±4.3%, n=12, P<0.05) or tails (40.7±6.0%, n=92, P<0.05) were lower than those of morphologically normal spermatozoa (61.6±5.3%, n=101). The follicular-fluid-induced [Ca2+]i increase of morphologically normal spermatozoa from infertile couples (35.1±6.3%, n=25, P<0.05) was also found to be lower than that of morphologically normal spermatozoa from healthy donors. Conclusion: The present study shows that spermatozoa with abnormal morphology in healthy donors have disorders of signal transduction, as do normally shaped sperm in men from infertile couples. (Reprod Med Biol 2008; 7: 143-149).