Sheep blood (SB) has been well recognized as a standard blood supplement in blood agar (BA) preparation. Since it is expensive and inconvenient to obtain, expired human blood (HuB) from local blood banks is commonly used to prepare blood agar in the routine microbiology laboratory in many developing countries including Thailand. The major problem of human blood agar (HuBA) is misdiagnosis, especially in streptococci whose identification is based on true hemolysis. This may be due to the different size of sheep and human red blood cells, as well as to inhibitors in human serum that affect hemolysis. Developing a technique to prepare HuBA of equal quality to sheep blood agar (SBA) would be useful. HuBA for culture and isolation of streptococci and other medically important bacteria was prepared from washed and unwashed red blood cells at concentrations of 3-5% vol/vol. Bacterial growth characteristics, e.g. colony morphology, hemolytic pattern and colony count were evaluated in comparison to SBA. It was found that a concentration of 3% HuB in the preparation of HuBA showed results equivalent to SBA. Specialized identification tests, including CAMP test and satellite test were performed with 3% HuBA. HuBA prepared from washed cells produced correct results in the CAMP and satellite tests, possibly because serum inhibitors were removed in the wash step. Therefore, our study indicates that 3% washed-HuBA can replace SBA in the routine laboratory in developing countries and constitutes a ready available alternative at significantly reduced costs. Keywords: Blood agar, hemolysis, culture, CAMP test, satellite test.