Case series summary This case series describes the clinical utility of antigen testing for the diagnosis of feline ocular histoplasmosis. Four cats with suspected (n = 2) or confirmed (n = 2) ocular histoplasmosis are described: three from Oklahoma and one from California. In one case, serial urine antigen tests, as well as a serum antigen test for Histoplasma capsulatum, were negative; however, light microscopy identified microorganisms consistent with H capsulatum in ocular tissues at necropsy. In a further two cats with recurrent ocular histoplasmosis following long-term systemic antifungal therapy, Histoplasma species urine antigen concentrations were negative, but both cats improved clinically following systemic antifungal therapy and remained in apparent clinical remission after treatment cessation (9-16 months). The final cat displayed profound bilateral endophthalmitis; however, Histoplasma species antigen testing of vitreous humor and subretinal fluid from the left eye was negative. Intralesional organisms were detected on histopathology of both eyes, and H capsulatum was subsequently isolated and sequenced from tissue of one eye. Relevance and novel information These cases highlight the potential difficulty in definitively diagnosing ocular histoplasmosis in cats when conducting antigen testing of serum, urine and even ocular fluids. Although antigen testing has previously proven useful in the diagnosis of disseminated feline histoplasmosis, it may not be adequate in cats with only ocular signs.