A ruminant mammary tissue culture that accurately reproduces mammary function in vitro would be a valuable tool in studies of ruminant lactation. This study was conducted to establish an in vitro model for culture of lactating bovine mammary tissue. The tissue still maintained the activity at 72 h, which was indicated by the value of MTT and oligonucleosomal fraction of cellular DNA. Under the conditions used in this study, the gene expression of αs1 casein was highest at 8 h, and then declined at 12 and 24 h. The results suggest that this primary tissue culture system may be useful in studying the effects of different combinations of amino acids on milk protein in terms of αs1 casein in vitro.