Differential hybridization was used to isolate genes potentially involved in the process of metastasis. Ten complementary DNAs (cDNAs) that were differentially expressed between a highly metastatic (MTLn3) and a nonmetastatic (MTC.4) line of the rat 13762NF mammary adenocarcinoma were isolated and sequenced. Examination of the EMBL/GenBank database revealed that one of the genes had a high degree of homology (98.8%) to annexin I (also known as calpactin II). Quantitative analysis of Northern blot hybridizations showed that the annexin I-like sequence was expressed 4- to 7-fold higher in MTLn3 than in MTC.4 cells. Steady state mRNA levels were also low in MTLn2, a cell line of low metastatic potential closely related to MTLn3, but were not related to metastatic potential in colon adenocarcinoma or melanoma cells. Two of the cDNAs (designated 8.11 and 10.14) were found to be novel. The expression of 10.14 mRNA (3.2 kb) was 4-fold higher in MTLn3 than in MTC.4 cells. Sequencing of the 10.14 cDNA (2.2 kb) revealed a putative open reading frame of 583 amino acids that was also novel. Expression of 8.11 mRNA (> 7 kb) inversely correlated with metastatic potential. Another differentially transcribed gene was highly homologous to ERK2 (extracellular signal related kinase 2), a mitogen-activated protein kinase (MAPK). Northern analysis of ERK2 expression revealed 3-fold higher amounts of a 1.3 kb mRNA in MTLn3 than in MTC.4 cells. Higher levels of ERK2 mRNA were generally seen in the more metastatic human colon but not in melanoma cell lines. We also corroborated the work of Taniguchi (Nucl Acids Res 19:6949, 1991) by independently identifying EF-1 alpha as a putative metastasis-associated gene.