Serum from wild alligators was collected and tested for antibiotic activity against three enveloped viruses using cell-based assays. Alligator serum demonstrated antiviral activities against human immunodeficiency virus type 1 (HIV-1; IC50=0.9%), West Nile virus (WNV; IC50=4.3%), and Herpes simplex virus type 1 (HSV-1; IC50=3.4%). The inhibitory concentration (IC50) is defined as the concentration of serum that inhibits 50% of viral activity. The antiviral effects of the alligator serum were difficult to evaluate at high concentrations due to the inherent toxicity to the mammalian cells used to assay viral activities. The TC50 (serum concentration that reduces cell viability to 50%) values for the serum in the HIV-1, WNV, and HSV-1 assays were 32.8, 36.3 and 39.1%, respectively. Heat-treated serum (56 degrees C, 30 min) displayed IC50 values of >50, 9.8 and 14.9% for HIV-1, WNV and HSV-1 viruses, respectively. In addition, the TC50 values using heat-treated serum were substantially elevated for all three assays, relative to untreated serum (47.3 to >50%). Alligator serum complement activity has been shown to be heat labile under these conditions. HIV-1 antiviral action was heat-sensitive, and thus possibly due to the action of serum complement, while the anti-WNV and anti-HSV-1 activities were not heat labile and thus probably not complement mediated.