The aim of this study is to determine the pharmacokinetics of tylosin and tilmicosin in serum and milk in healthy Holstein breed cows (n=12) and reevaluate the amount of residue in milk. Following the intramuscular administration of tylosin, the maximum concentrations (Cmax) in serum and milk were found to be1.30±0.24and4.55±0.23 µg/mL, the time required to reach the peak concentration (tmax) was found to be 2nd and 4th h, and elimination half-lives (t1/2β) were found to be20.46±2.08and26.36±5.55 h, respectively. Following the subcutaneous administration of tilmicosin, theCmaxin serum and milk were found to be0.86±0.20and20.16±1.13 µg/mL, thetmaxwas found to be 1st and 8th h, and thet1/2βwere found to be29.94±6.65and43.02±5.18 h, respectively.AUCmilk/AUCserumandCmax-milk/Cmax-serumrates, which are indicators for determining the rate of drugs that pass into milk, were, respectively, calculated as5.01±0.72and3.61±0.69for tylosin and23.91±6.38and20.16±1.13for tilmicosin. In conclusion, it may be stated that milk concentration of tylosin after parenteral administration is higher than expected like tilmicosin and needs more withdrawal period for milk than reported.