OBJECTIVE To compare polyacrylamide gel as synthetic medium with human cervical mucus (CM) for the in vitro sperm-penetration test during infertility investigation. METHODS One hundred sixty-nine randomly chosen couples with a median duration of infertility of 4 (range, 1 to 16) years presenting at the infertility unit of the Women's University Hospital of Heidelberg, Germany. METHODS Evaluation of sperm migration in polyacrylamide gel used in four different concentrations (1.5%, 1.6%, 1.7%, 1.8%) in the capillary tube test in parallel with CM of patients' female partners and CM of fertile donors, obtained under standardized conditions. Correlation of migration test results with outcome of semen analysis including microbial cultures and testing for local antisperm antibodies by means of the mixed antiglobulin reaction, postcoital testing, and the subsequent pregnancy rate after control for female infertility factors in a prospective study. RESULTS Sperm ability to penetrate the synthetic medium (concerning all concentrations) correlated significantly with the penetration of human CM, although polyacrylamide proved to be a stronger barrier. Sperm velocity and duration of progressive motility were markedly reduced in polyacrylamide. Polyacrylamide results correlated with the outcome of standard sperm analyses but not with sperm antibody testing. No clear differentiation was obtained with regard to subsequent fertility (19% after 6 months), although adequate sperm migration in polyacrylamide 1.8% was significantly more frequent in the fertile group. CONCLUSIONS In analyzing the intrinsic motility, penetration testing with polyacrylamide gel provides important information not obtained by routine sperm analysis. However, particularly with regard to immunological factors and fertility prognosis, human CM should be preferred whenever possible.