Abstract: Background: Mother-to-child transmission of HIV is the most significant route of HIV infection in children and by far the largest source of HIV infection in children below the age of 15 years. Objectives: the aim of this study was to assess current practices and future intention of lactating mothers on VCT and infant feeding in relation to prevention of MTCT of HIV in DURH. Methods: Institutional based cross sectional quantitative survey was conducted from February, 1 to February 10 2008 E.C using standardized questionnaire, among lactating mothers in DURH selected using probability proportional to size. Non probability conveniences (purposive) sampling technique was used .The data was exported in to SPSS software version 20 and analyzed. Official written letter of cooperation were secured from DURH to the concerned body, and verbal consents of the study participants was obtained. Finally the result was presented by using tables and charts. Result: A total of 271 (100%) mothers participated in the study. The majority of the study participants were in the age group of 25 to 34 (61.8%) years. The majority were married 219 (80.8%), house wives in occupation, 113 (41.7%), and had Grade 7-12 that 69 (25.5%) attended high school education. There was statistically association between knowledge and the respondents’ educational status (p=0.037), association between attitude towards VCT and the respondents’ educational status (p=0.010), as well as between attitude towards VCT and occupation (p=0.021) and there was statistically association between practice towards VCT and the respondents’ religion (p=0.019), as well as between practice towards VCT and monthly income (p=0.041). Conclusion and Recommendation: The findings from this study in relation to prevention of HIV transmission revealed that mothers had good knowledge of HIV/AIDS and on MTCT and PMTCT. The study participants also have good attitude towards VCT which may have a contribution to intention of the mothers to have HIV test. Therefore to increasing utilization of PMTCT, infant feeding and VCT it requires to address reinforce the importance of exclusive breast feeding during antenatal and postnatal follow up, prevention of mother to child transmission of HIV (PMTCT) service shall be accessed to some groups of the community such as housemaid and housewives as much as possible to get information utilize the services, and establish communication/ referral network between health facilities and organizations working on HIV/AIDS.