Two human cell lines, one established from a colon carcinoma (SW480) and the other from its lymph node metastasis (SW620), were compared with respect to their migration capacity employing a three-dimensional collagen matrix and time-lapse video recording. Non-motile cells were characterized by a round shape, whereas motile cells appeared in an elongated form with pseudopodia. The primary tumor cells showed a higher spontaneous locomoting activity than the cells from the metastasis. Using single cell analysis, the distance migrated within 15 h was slightly increased in the presence of hyaluronic acid (HA) in both cell lines. An investigation of the amount of CD44 on the cell surface using the anti-CD44 antibody Hermes-1 showed only minor concentrations of this glycoprotein on cells from the metastasis, whereas a much higher amount was found on cells derived from the primary tumor. The distribution of CD44 on the cell surfaces of HA-treated and untreated cells did not differ as shown by confocal laser scanning microscopy in SW480. The results indicate a restricted influence of HA on migration in the two cell lines.