In malignant tumors, metastasis genes are typically deregulated by aberrant expression or splicing. Osteopontin is expressed at high levels by various cancers and contributes importantly to their invasive potential. In contrast, osteopontin derived from host cells induces cellular immunity and could bolster antitumor protection by cytotoxic T lymphocytes. Here we show that breast cancer cells express multiple splice variants of osteopontin. According to RT-PCR analysis of human breast tissue specimens, the splice variant osteopontin-c is a highly specific marker for transformed cells, which is not expressed in their surrounding normal tissue. The full-length form of osteopontin aggregates in the presence of physiologic amounts of calcium and, in this state, leads to enhanced cell adhesion. Ostensibly, this effect is inhibitory for tumor cell dissemination. The shortest splice variant, osteopontin-c, does not aggregate in the presence of calcium and enhances clone formation in soft agar. According to microarray analysis, osteopontin-c induces the expression of oxidoreductases, consistent with protection from anoikis during anchorage-independent growth. These studies define a third functional domain of osteopontin, beside the C-terminal CD44-binding site and the central integrin-binding site. They also provide evidence for a bifunctional character of osteopontin, with the soluble form supporting invasiveness and the aggregated form promoting adhesion.