Five new steroid sulfates, sodium 2beta,3alpha-dihydroxy-5alpha-cholestane 3-sulfate (6), sodium 2beta,3alpha-dihydroxy-5alpha-cholestane 2-sulfate (7), disodium 2beta,3alpha-dihydroxy-5alpha-cholestane disulfate (8), sodium 3alpha-acetoxy-2beta-hydroxy-5alpha-cholestane 2-sulfate (12), and sodium 2beta-acetoxy-3alpha-hydroxy-5alpha-cholestane 3-sulfate (13), have been synthesized starting from 3beta-hydroxy-5alpha-cholestane (1). The synthetic steroids were completely characterized by one-dimensional and two-dimensional NMR and FABMS spectra. Sulfation was performed using triethylamine-sulfur trioxide complex in dimethylformamide as the sulfating agent. The sulfated steroids were comparatively evaluated for their inhibitory effect on the replication of herpes simplex virus type 2 (HSV-2). Compounds 7 and 8 were the most effective in their inhibitory action against HSV-2. The disulfated steroid 8 also proved to be active against DEN-2 and JV.