Background and Objective: People who use drugs (PWUD) are at high-risk for hepatitis C virus (HCV) infection. Opiate substitution treatment (OST) reduces the risk of infection. Testing opportunity acceptance and seroconversion rates among PWUD under OST are of great importance for public health. Methods: AntiHCV rapid test was offered free of charge to all individuals under OST in Athens (N=2,892) from Sep2021 to Oct2022. A retrospective analysis of data collected was conducted. In total, 83.3% were male, mean (SD) age 51(9) years, 56.3% buprenorphine/43.7% methadone, in OST public units of large(>200patients:35.3%), medium (100-199patients:31.5%) or small capacity (<99 patients:33.2%). Results: From 2,892 participants 2040(70.5%) have a previous positive test and were not retested. AntiHCV test was performed to 540 out of 852 eligible persons (63.4%); Of the 312 not tested, 42(13.5%) refused screening, 8(2.6%) were temporarily out of OST and 262/312(84%) did not present for testing due to unknown reasons. Tested individuals didn’t differ significanlty with those who refused testing with respect to gender, age and opiate substitution substance. Refusal rate was slightly higher in medium-capacity units (19.9%) vs smaller (14.9%) and larger (13%) ones, however the differences were not statistically significant. No significant differences were observed among patients who refused testing and the rest not screened. In total 103/540(19.1%) were found AntiHCV(+). Seroconversion, defined as a positve rapid test in a known antiHCV (-) individual, was documented in 98/540(18,1%) and it was higher in large-capacity (24.3%) compared to medium-capacity units (6.9%) (p<0.001). Seroconversion rate was higher in methadone participants (21.3%) vs buprenorphine (15.4%), although the difference was not statistically significant. Rates didn’t differ significantly by sex and age. Conclusions: High rates of antiHCV seroconvertion are documented among PWUD even when under OST. Further study on high-risk behaviors while under substitution is needed. Testing acceptance by PWUD is a challenge even when test is offered free of charge in a treatment setting.