In vitro sperm migration assays were performed using bovine spermatozoa and cervical mucus. Experiments were designed to test the effects of storage temperature, method of storage, duration of storage, and source of cervical mucus. Significant variation in migration of spermatozoa was due both to differences in mucus samples and to short-term mucus storage at temperatures ranging from ambient to -196 degrees C. The parallel-orienting effect of cervical mucus on migrating sperm was shown to be a major factor in quantitative assays based upon migration distance. Thus, comparisons of migration among different specimens of semen likely will be biased unless the tests are run simultaneously. Implications of these results are discussed relative to the performance of quantitative sperm migration assays in the clinical or research laboratory.