Modifications of the usual assay methods for nicotinic acid, biotin, and pantothenic acid, which were introduced in applying them to cow's milk samples, have been described. There was no evidence that a bound form of any of these vitamins exists in milk. The biotin levels showed great variation among the milks of individual cows. Individual averages for eight cows ranged from 11 to 37 μg per liter. Between samples obtained from the same cow on successive days, the variation was also large. These variations were less marked in the case of nicotinic and pantothenic acids. The nicotinic acid content was found to decrease during the winter and early spring months, and to increase during the summer and early fall months. The range of values on May 9 for eight cows was 0.22 to 0.77 μg per ml. On October 16 for five cows it was 0.56 to 0.96 μg per ml. A possible correlation with pasture feeding has been suggested. The biotin content had no apparent relation to the season. The concentration of nicotinic acid in the milk decreased regularly through much of the lactation period. The pantothenic acid content of the colostrum at the time of freshening was found to be relatively low, the average level being about 1.5 μg calcium pantothenate per ml. The mean then rose during the next 9 days to a maximum of about 4 μg per ml, and then dropped to a normal level at about 3.5 μg per ml. A similar, though earlier, rise to a maximum and fall to normal tends to be true of the biotin level of milk. The pantothenic acid and biotin contents of milk are correlated, though the nicotinic acid level appeared not to be correlated with either. All three vitamins were found to be stable during pasteurization and exposure of the milk to sunlight.