The incidence of testicular cancer is rising. Despite a high cure rate, efforts should be made to obtain diagnosis at the pre-invasive intratubular carcinoma in situ (CIS) stage, as the disease is potentially lethal and treatment has severe side-effects, especially regarding reproductive function. CIS diagnosis is presently only possible by a surgical biopsy of the testis. Immunocytological staining for transcription factor activator protein (AP-2γ), previously identified as a marker for neoplastic germ cells, was performed in centrifuged samples of ejaculates obtained from 104 andrological patients, including patients with testicular cancer and subfertility. Cells positive for AP-2γ were found only in semen samples from patients diagnosed a priori with testicular neoplasms and, surprisingly, in a 23 year old control subject with oligozoospermia and no symptoms of a germ cell tumour. Testicular biopsies performed during the follow-up of this patient revealed widespread CIS in one testicle, thus proving a potential diagnostic value of the new marker. For the first time, a patient without clinical symptoms of testicular neoplasia was diagnosed at the pre-invasive CIS stage using a new, simple method based on immunocytological staining of a semen sample for AP-2γ, a novel marker for CIS. The value of this method for diagnostic use in the clinic requires further careful validation in a large series of patients and controls, but the preliminary results are promising.