Insulin-like growth factor II (IGF-II) is expressed in many developing embryonic tissues and is involved in mammalian growth and development. After birth, serum IGF-II is mainly produced by liver cells. Many reports have indicated that IGF-II is overexpressed in some hepatocellular carcinoma (HCC) tissue. These findings imply the possible importance of this growth factor in carcinogenesis. We screened four human HCC cell lines and three rat HCC cell lines and found that HuH-7 and HepG2 cells produced fivefold more intracellular IGF-II than the other cell lines. Experimental data indicate that IGF-II functions through the intracrine mode for HuH-7 cells. To study whether the overexpression of IGF-II is significant for the growth of HCC or only a consequence of HCC development, we used antisense oligodeoxynucleotides (ATON) to arrest the translation of IGF-II mRNA, and then measured the effects on cell growth. We found that the production of IGF-II was suppressed by ATON, and the decrease of IGF-II resulted in growth inhibition of HuH-7 and HepG2. ATON had no effect on the other tested cell lines, which produced lower levels of IGF-II. The growth inhibition was mainly attributed to a decrease of cell proliferative activity. The results indicate that the IGF-II-overproducing cell lines do depend on IGF-II for growth, and ATON of IGF-II can selectively inhibit the growth of these cells. ATON may be a potential therapeutic agent for this type of HCC in vivo.