By immunodiffusion assay (ID-test) milk samples of mice of several strains and of F1-hybrids of the GR strain were tested for the presence of mammary tumour virus (MTV) antigens. The results clearly demonstrated that the presence of viral antigens in the milk of the first lactation period is restricted to mice harbouring endogenous MTV-GR. Viral antigens were detectable in about 50% of the milk samples collected during the first (occasionally the second) lactation periods of mice of the segregating backcross I (Bc I) populations: DBAfX(DBAfXGR), AKRX(AKRXGR), BALB/cX(BALB/cXGR) and C57BLX(C57BLXGR), indicating that one dominant gene is responsible for the presence of viral antigens in the milk of GR mice. The proposed gene symbol is Mtv-2. Milk samples from female mice of three different Bc II populations were tested for the occurrence of viral antigens. In the first Bc II: [BALB/cX(BALB/cXGR)]XBALB/c 33 out of 51 mice, descending from ID-positive mothers, had ID-positive milk and only one out of 71 mice, which were the progeny of ID-NEGATIVE Bc I mothers, was ID-positive. These results may be influenced by the MTV transmitted extrachromosomally via the milk of the mother. The two other Bx II populations were derived from Bc I fathers, either [BALB/cX(BALB/cXGR)] or [(BALB/cXGR)XBALB/c] f and BALB/c females. The results obtained with these Bc II populations suggested that 6 Bc I fathers were heterozygous for Mtv-2. Since the segregation ratio (60:29) in the Bc II population (progeny of these 6 Bc I male) deviates significantly from the expected 1:1 ratio, one may assume extrachromosomal transmission of MTV via the seminal fluid of the father to the progeny. A close correlation was found between the presence of MTV antigens in the milk and the occurrence of both early mammary tumours after hormone treatment and spontaneous mammary tumours before the age of 13 months. These results suggest that the early appearance of mammary tumours in the GR strain and the early expression of MTV antigens in the milk appear to be controlled by the same genetic factors.