After occupational exposures, immediate HIV testing of source patients may avoid the unnecessary use of post-exposure prophylaxis (PEP). Two time periods were compared. Before the availability of 24 h a day immediate testing, PEP was initiated after 12.6% of exposures, compared with 3.7% during the second period. The adjusted relative odds ratio of PEP during the second compared with the first period, was 0.23. The availability of immediate HIV testing limits unnecessary occupational PEP.