Hoechst 33342 in vivo staining was combined with immunofluorescent staining of cell surface antigens to quantify the distribution, relative to blood supply, of lymphocytes in a preneoplastic mammary lesion, the murine C4 hyperplastic alveolar nodule (HAN), and the C4 adenocarcinoma which develops from C4 HAN. The vascular supply to lymphocytes expressing Thy 1.2, L3T4, Ly2, and ASGM1 cell surface antigens was evaluated in both tissues. The distribution of ASGM1+ cells, which include natural killer cells, differed between the two tissues, being significantly increased in the 20% brightest Hoechst-stained lymphocyte fraction in HAN but not in C4 tumor. Distribution of T lymphocytes did not differ between the two tissues. The combination of in vivo Hoechst 33342 with in vitro immunofluorescence provides a simple method to evaluate the distribution with regard to blood supply of lymphocyte subsets in solid tumors and preneoplastic lesions.