In the five-year period between 1989 and 1993, 87 needlestick accidents occurred among healthcare workers at our hospital. Thirty-seven (43%) of these needlestick accidents involved blood contaminated with hepatitis C virus (HCV), and two of them (5.4%) led to the occurrence of hepatitis C infection. Case 1 was a 43-year-old nurse who was accidentally injured by a needle contaminated with blood from a patient who had cirrhosis and hepatocellular carcinoma due to hepatitis C. Acute hepatitis C occurred after five weeks and HCV RNA was positive after eight weeks. Case 2 was a 33-year-old nurse who was injured by a needle contaminated with blood from a patient who had chronic hepatitis C. Liver function was normal at 11 days after the accident. However, hepatitis C was diagnosed 21 months later after she had successfully given birth to her baby. The nucleotide sequence of the HCV E2/NS1 region was determined in the two patients and the needlestick victims, and phylogenetic trees were constructed by molecular evolutionary analysis. On the basis of these trees, transmission of HCV could be confirmed in both cases. This method of analysis may be useful for confirming the transmission of HCV even long after the event.