Four lactating Holstein cows (average milk yield: 20 +/- 3 l/d) were used to develop and validate a method for estimating the size of udder cisterns (Sinus lactiferi) using ultrasonography. A sectorial transducer probe of 5 MHz, placed in contact with the teat in a parallel cranial position, was used to obtain vertical scans of the udder in two perpendicular planes with the teat canal axis as reference. Udder scans for each udder quarter were taken randomly at intervals of 4, 8, 12, 16, 20 and 24 h after milking. Glandular parenchyma (echogenic) and lumen of the cisterns full of milk (anechogenic) were evident in the scans, the calculated area of the anechogenic portion being defined as cistern area. Cistern areas measured in perpendicular scans were highly correlated. Immediately after each measurement, cisternal milk was removed from each quarter using a teat cannula after i.v. injection of an oxytocin-receptor blocking agent. Alveolar milk from each quarter was then obtained by machine milking after i.m. injection of oxytocin. Cistern area and cisternal milk volume increased with length of milking interval showing a curvi-linear pattern with a plateau after 16 h. Correlations between cistern area and cisternal milk volume were positive and significant (P<0.001) at all intervals but showed the highest values with the smallest residual standard deviations at 8 h (r=0.88) and 12 h (r=0.84). Since 8 h has previously been identified as a suitable time at which to determine cisternal milk volume for the purposes of defining suitability for different milking strategies, we conclude that ultrasonography provides a satisfactory, non-invasive method for determination of milk storage characteristics in dairy cows.