Mammary tumors have ceased to develop in descendants of one female of the RIII/AnOk mouse strain which was brought from the Mouse Colony of Okayama University Medical School in 1975, while descendants of another female of this strain have maintained a high tumor incidence. The former and latter descendants were separated out as RIII/AnOk/2 and RIII/AnOk/1 sublines, respectively. The amount of mouse mammary tumor virus (MMTV) in the milk of individual females in these two sublines was determined by means of a sensitive enzyme immunoassay. No MMTV antigens were detected in 59 milk samples of RIII/AnOk/2 females collected during the 1st to 7th lactation periods. On the other hand, all the milk samples of RIII/AnOk/1 females contained MMTV antigens with concentrations ranging from 9 to 400μg/ml. When RIII/AnOk/2 mice were foster-nursed by RIII/AnOk/1 mothers, the infected RIII/AnOk/2 females produced MMTV in the milk to almost the same degree as did the RIII/AnOk/1 females. No genetic differences between the two sublines were observed by the use of reciprocal skin grafting, tumor transplantation, and analysis of biochemical genetic markers. These results indicate that the arrest of mammary tumor development in the RIII/AnOk/2 subline was due to the disappearance of MMTV in the milk.