Males of the MT-PVLT-10 transgenic mouse line, which expresses the polyomavirus large T-antigen under the control of the metallothionein promoter, develop testicular adenomas and display seminal vesicle enlargement. Histological analysis of adenomatous testis indicates a predominance of Leydig cells, with few normal Sertoli cells or seminiferous tubules remaining. Primary cell cultures established from the testes of control nontransgenic animals (all ages) and young MT-PVLT-10 animals (before the appearance of any phenotype) quickly entered crisis and died. In contrast, permanent cell lines could be derived from pre- and postadenomatous testes from older MT-PVLT-10 mice. All primary cultures and cell lines expressed large T-antigen. A primary culture (D-37) derived from an MT-PVLT-10 male with normal testes but enlarged seminal vesicles has been maintained for over 2 years and experiments indicate that the D-37 culture is unable to form tumors in nude mice. In contrast, a primary culture (D-4) derived from adenomatous testes of an MT-PVLT-10 mouse is also immortal, but injection of this culture into nude mice consistently resulted in tumor formation. Cloning of the D-4 culture resulted in pure Sertoli or Leydig cell clones, neither of which could form tumor upon injection into nude mice. Injection of a mixture of both cell types did result in tumor formation, suggesting a dynamic interaction between these cell types in MT-PVLT-10-induced tumorigenesis.