Chronic nonbacterial prostatitis spontaneously develops in aged Lewis and Wistar rats but not in Sprague-Dawley rats. We report the unique profile of lymphocyte subsets present in the healthy Sprague-Dawley rat prostate. We compared enzymatic and mechanical methods of intraprostatic lymphocyte isolation in healthy 8 to 10-week-old Sprague-Dawley rats. Mechanical isolation was chosen because of its superior preservation of surface antigens. Intraprostatic lymphocyte subsets were analyzed by flow cytometry. Levels of prostatic αβTCR+ T cells were similar and levels of prostatic B cells were decreased 5 to 10-fold compared with those found in other tissues (p ≤0.005). Unexpectedly two-thirds of the total prostatic lymphocytes expressed the natural killer (NK) marker CD161a+. They were divided equally between NK (CD161a+αβTCR − ) and NKT (CD161a+αβTCR+) cells. Of prostatic CD161a+ cells 50% to 60% were also CD8+. Levels of NKT cells were dramatically lower in other tissues (p ≤0.005) and they never accounted for more than 8% of total lymphocytes. The prostate contained lower levels of CD4+ T cells than all tissues except the liver (p ≤0.005) and higher levels of CD8+ T cells than any other tissue studied (p ≤0.05), resulting in an inverted CD4-to-CD8 ratio. CD45RC+CD4+αβTCR+ T cells and CD161a+CD4+αβTCR+ NKT cells were elevated in the prostate (p ≤0.02). The healthy rat prostate contains an unusually high proportion of NK and NKT cells. The balance between the CD45RC+CD4+αβTCR+ T cells, which initiate the cell mediated immune response, and CD4+ NKT cells, which can suppress autoimmunity, may be a key in understanding the resistance of Sprague-Dawley rats to chronic nonbacterial prostatitis.