Objective : To assess the effects of progesterone and acetyl-L-carnitine used after treated with Isolate?? gradient before semen cryopreservation-thawing on sperm parameters and membrane integrity. Material and Methods : From April 2001 to July 2001, ten normal male partner of couples who were visited in vitro fertilization (IVF) clinics. the semens were treated with $Isolate^{(R)}$ gradient before cryopreservation, spermatozoa was incubated with progesterone (1, 5 and $10{mu}M$), acetyl-L-carnitine (2.5, 5 and $10{mu}M$), or both (progesterone, $1{mu}M$; and acetyl-L-carnitine, $5{mu}M$) for 30 min. Results: There were no differences in sperm parameters and vital stain among isolate only treated group, progesterone (1, 5 and $10{mu}M$), acetyl-L-carnitine (2.5, 5 and $10{mu}M$) and both (progesterone, $1{mu}M$; and acetyl-L-carnitine, $5{mu}M$). But, in high concentration of acetyl-L-carnitine ($10{mu}M$) treated group, sperm parameters and vital stain were decreased. The statistical method was used ANOVA (Kruskal-Wallis test) and p value was <0.01. Conclusions : Neither progesterone nor acetyl-L-carnitine show to be protective effect on the cryodamage assessed by sperm parameters and vital stain (eosin-Y stain) in normal sperm. High concentration of acetyl-L-carnitine ($10{mu}M$), however, was harmful effect on cryoprevention.