Herpes simplex virus (HSV) types 1 and 2 are widespread human infectious agents that are responsible for persistent and latent infections. HSV type 2 (HSV-2) infection is usually transmitted sexually, while HSV type I (HSV-1) is commonly acquired by saliva contact during childhood. In a retrospective study, sera from more than 4,800 patients were analyzed for HSV type-specific IgG antibodies. In people older than 15 years, the seroprevalence of HSV-1 showed no statistically significant discrepancy between the control group (76.3% in females and 75.2% in males), HIV-infected patients (82.8% in females and 84.3% in males), and organ transplant (OTX) recipients (90.3% in females and 86.3% in males) (P>0.05). Age-related analysis of the control group showed that there is an age-dependent increase of HSV-1 seroprevalence in both sexes, reaching its peak in those aged 40 years and older (women 85.4%, men 82.8%). The only age group in which there is a significantly higher seropositivity rate in women than in men is in those aged between 15 and 39 years, with 70.8% versus 63.7% (P<0.05). As with HSV-1, there is an age-related increase of the HSV-2 seroprevalence; however, this increase starts later in life, with the onset of sexual activity. The HSV-2 prevalence across all age groups was highest in female prostitutes (78.0%) and among HIV-infected patients (women 64.1%, men 54.3%); this contrasts with the control group (overall women 17%, men 12.5%; those above 15 years of age, women 18%, men 13.8%) and the OTX patients (women 22.6%, men 9.8%). In the control group the rate of positivity increases with age and peaks in the group older than 40 years (24.2% in women and 16.2% in men). In females the seroprevalence is always elevated compared with males. The data presented show that female sex and older age are independent predictors of HSV-2 seropositivity, while immunosuppression is not. Our additional data show no evidence of a statistically significant humoral HSV-1/HSV-2 cross-immunity. People with HSV-1 serum antibodies have no lower risk of HSV-2 seropositivity than those lacking antibodies to HSV-1. The same is true when investigating HSV-1 seroprevalence rates in HSV-2-seropositive or -negative individuals, retrospectively.