The whole plant of Justicia procumbens L. (Acanthaceae), known as "Chu Wei Hung" or "Ju Chuang" in Chinese folklore, is used as an herbal remedy for the treatment of fever, pain due to pharyngo-laryngeal swelling, and cancer. Prior phytochemical studies on this plant have afforded justicidin-C and justicidin-D. As a result of continuing searches among Chinese medicinal plants for novel, naturally occurring potential antitumor agents, the methanolic extract of the whole plant of J. procumbens was found to show significant inhibitory activity in vivo against P-388 lymphocytic leukemia growth in BDF1 male mice (T/C=150% at 50 mg/kg/day, IP) as well as in vitro cytotoxicity in the 9-KB (human nasopharyngeal carcinoma) cell culture assay. Bioassay-directed fractionation of the MeOH extract led to the isolation of five 2,3-naphthalide lignans, which were identified as justicidin-A (1), diphyllin (2), neojusticin-A (i.e., justicidin-D) (3), neojusticin-B (i.e., justicidin-C) (4), and justicidin-E (5) by spectral analysis and direct comparison with authentic samples of the first four compounds as well as data for the last compound reported in the literature. Compounds 1-4 were previously isolated from J. procumbens var. leucantha. Compound 1 was also reported as a fish-killing component of Justicia hayatai var. decumbens. A comparison of the cytotoxicity (KB) clearly indicated that the γ-lactone ring carbonyl α to C-3 instead of C-2 is required for potent cytotoxicity. Thus, 1 and 2 were active (ED50 (KB)<1.0 μg/ml each), while 3-5 were inactive (ED50 (KB)=9.0 μg/ml for 3 and >10.5 μg/ml for 4 and 5 each).