We have previously recovered herpes simplex virus type 1 (HSV) from the corneas of latently infected mice by cultivation in vitro. It could be argued, however, that these data do not definitively distinguish between persistent and latent corneal infection. We have now used RNA hybridization in situ to resolve this question by determining the expression of HSV genes in the corneas of BALB/c mice during latent infection. Two to four months after topical corneal inoculation with HSV, when no active ocular disease or infectious virus was present, corneas were removed and digested with collagenase. Dissociated cells pooled from two corneas were hybridized with 3H- or 35S-labeled 2.6-kb single-stranded RNA probes to detect sense and antisense ICP-0 transcripts. Twenty-five percent of the pools hybridized with the probe for antisense ICP-0 (latency-associated transcript, LAT), while only 3% hybridized with the probe for ICP-0 (p less than 0.03). Of the cells in positive pools, 0.6-7.0% showed a positive hybridization signal for LAT. No infectious virus was found by culture of supernatants from the probed pools or control latently infected corneas. These data provide further evidence that HSV can establish a true latent infection in the mouse cornea.