We have examined tumor progression and metastatic properties of three clonal murine mammary tumor cell lines of recent origin (D2A1, D2.OR and D2.1). These lines were derived from spontaneous mammary tumors which originated from a D2 hyperplastic alveolar nodule (HAN) line. D2A1 cells were more malignant than D2.OR or D2.1 cells, whether measured by experimental metastasis assays after intravenous injection in nude mice or chick embryos, in vivo growth rate of primary tumors following mammary fat pad injection in nude mice, or spontaneous metastasis assay from primary tumors growing in mammary fat pads. D2A1 cells also were more invasive in vitro in a Matrigel invasion assay than D2.1 cells, while the D2.OR cells were non-invasive in this assay. The increased invasiveness and malignancy of D2A1 cells were associated with increased levels of mRNA for the cysteine proteinase cathepsin L. Levels of osteopontin (OPN), nm23, int-1 and int-2 mRNAs were also examined. Nm23 levels were highest in the most malignant cell line. These cell lines provide a model for studying the tumorigenic and metastatic ability of mammary tumor cells and offer several advantages: they were cloned from mammary tumors that originate from a common source of preneoplastic cells (D2HAN); they are of relatively recent origin; and they have spontaneously arrived at different stages of tumor progression.