We have previously reported sex- and estrous-related differences in host resistance to the metastatic development of a mammary adenocarcinoma cell line, MADB106, in the Fischer 344 (F344) rat. In other studies, we found that surgery suppressed natural killer (NK) cell activity and increased the NK-sensitive metastatic development of MADB106 tumor cells. The current study was designed to explore whether sex or estrous phase at the time of surgery impacts the degree of such deleterious effects of surgery. Such estrous effects could be related to an ongoing clinical debate regarding the importance of the timing of breast cancer surgery with the menstrual cycle in premenopausal women. Mature F344 males and cycling females underwent either experimental laparotomy with halothane anesthesia, halothane anesthesia alone, or were untreated. Five hours after surgery, animals either were injected with radiolabeled MADB106 tumor cells and assessed for lung tumor cell retention 12 hours later, or underwent blood withdrawal for in vitro assessment of NK cell activity. MADB106 tumor cells metastasize only to the lungs, and lung tumor cell retention is: a) an early indicator of the number of metastases that would develop weeks later, and b) highly sensitive to in vivo levels of NK activity. This mammary adenocarcinoma cell line is syngeneic to the inbred F344 strain of rats used in our studies, thus constituting a model for breast cancer metastasis. The results indicated that sex, estrous phase, and surgery interacted in their effects on NK cell activity and tumor metastasis. MADB106 lung tumor cell retention was increased by surgery in both sexes (2- to 3-fold) compared to the anesthesia only and control groups. This increase, however, was significantly greater in proestrus/estrus (P/E) females than in metestrus/diestrus (M/D) females. Among the control animals, females in P/E exhibited significantly less NK cytotoxic activity compared to the males, and the NK activity exhibited by females in M/D was between these two groups. Surgery suppressed NK cytotoxic activity to a similar level in all groups. Possible implications of these findings for the surgical care of women with breast cancer are discussed.