We developed a fully automated three-dimensional cell tracking system that quantified the effect of extracellular matrix components on the infiltration and migration of tumor cells. The three-dimensional trajectories of two highly invasive cell lines, the human HT-1080 fibrosarcoma and the human MDA-MB-231 adenocarcinoma, were determined for long-term infiltration in plain or Matrigel-containing collagen type I gels. We modeled the trajectories with a novel formulation of the continuous Markov chain model that can distinguish between the tendencies for infiltration or lateral motion. Parameters such as the speed of subpopulations, the persistence of motion in certain directions, the turning frequency of the cells, the ultimate direction of motion, and the cell distribution with the infiltration depth were obtained to quantify the migration and infiltration at the cellular level. Distinct migratory and infiltration phenotypes were identified for the two cell types that were significantly dependent on gel composition. The HT-1080 cell line expressed a high motility phenotype on the plain collagen gel surface. The Matrigel-containing gel significantly enhanced the infiltration and the turning frequency of the HT-1080 cells. This study shows that tumor cell infiltration and migration are dynamic processes that depend significantly on the cell type and the microenvironment.