High-performance liquid chromatography was used for quantitative determination of podophyllotoxin in serum subsequent to repeated applications of a 0.5% ethanolic preparation on condylomata acuminata. The drug was not detectable in the serum of ten men treated with < or = 50 microliters. In serum of seven patients receiving 100-1,500 microliters on extraordinarily large en-plaque lesions, peak levels of 1-17 ng/ml were measured within 1-2 hr. The drug did not accumulate in serum. Dispersion of 100 microliters gave rise to peak levels of < or = 5.0 ng/ml and to subsequent levels of < or = 3.0 ng/ml for 4 hr. Larger doses gave rise to traceable amounts in serum for < or = 12 hr. Investigations with 52 patients indicated that volumes of > 100 microliters are rarely required for topical treatment of condylomata. It is concluded that application of volumes of < or = 250 microliters twice daily for three days would satisfy the most stringent safety precautions and avoid any danger of systemic toxicity. Potential hazards associated with use of the nonstandardized 20% podophyllin preparations are emphasized.