·AIM:We compared polymerase chain reaction(PCR)to cell culture isolation for the laboratory diagnosis of ocular herpes simplex virus(HSV)disease.·METHODS:Laboratory and medical records of consecutive patients were reviewed for results of(1)HSV PCR testing,(2)HSV cell culture isolation,and(3)clinical diagnosis.PCR results were statistically compared to cell culture isolation and patients initially diagnosed for ocular HSV infection.·RESULTS:Of 581 cases submitted for laboratory testing,520 were PCR negative,cell culture negative(89.6%);0 was PCR negative,cell culture positive(0%);27 were PCR positive,cell culture negative(4.6%);and 34 were PCR positive,cell culture positive(5.8%).PCR tested more positive than cell culture isolation(McNemar’s,P=0.0001).Of 47 HSV PCR positive cases with complete medical records,19 were cell culture negative for HSV and 28 were cell culture positive for HSV.Fourteen of 19 cell culture negative cases(74%)(Without PCR,5 cases of HSV would be missed)and 25 of the 28 cell culture positive cases(89%)(Laboratory testing was necessary for diagnosing 3 cases)were clinically diagnosed with HSV at the initial examination.·CONCLUSION:PCR was a more definitive test for diagnosing HSV ocular infection than cell culture isolation.Cell culture isolation alone can miss an atypical presentation of HSV ocular infection.·