The objective of this study was to determine if characteristics of sperm motion determined by computer-aided semen analysis (CASA) after thawing and preparation on discontinuous gradient could predict pregnancy outcome after intrauterine insemination (IUI) from frozen donor sperm. A retrospective analysis of 100 non-selected women undergoing 171 consecutive donor insemination cycles was conducted between January 2006 and April 2007. Semen samples from all donors were analysed after thawing and density gradient preparation. Women who became pregnant and those who did not were comparable in terms of age, ovarian stimulation regimen and indication of IUI with donor semen. Pregnancy rate per cycle was 21.8%, and pregnancy occurred after 2.5 IUI cycles on average. Motility parameters of sperm measured by CASA (VAP, VCL, VSL, LIN, STR, and ALH) and total spermatozoa concentration after preparation on discontinuous gradient showed no difference in both groups. Progressive and total motile spermatozoa concentration, as well as progressive and total motile percentages was significantly higher in pregnancy group. The receiver operating characteristic (ROC) curve analysis showed that total motile percentage >17% and motile concentration >0.9 x 10(6)/mL best predicted pregnancy. In a multivariate analysis, only total motility percentage was able to predict pregnancy. Sperm motility parameters of frozen-thawed prepared donor sperm obtained by CASA do not seem to predict pregnancy in IUI cycles. Total motile and progressive percentages and concentrations remain the best prognostic elements for pregnancy in IUI with donor semen.