This study determined the vitamin D(3) content and variability of retail milk in the United States having a declared fortification level of 400 IU (10 μg) per quart (qt; 1 qt=946.4 mL), which is 25% daily value per 8 fluid ounce (236.6 mL) serving. In 2007, vitamin D(3) fortified milk (skim, 1%, 2%, whole, and 1% fat chocolate milk) was collected from 24 statistically selected supermarkets in the United States. Additionally, 2% milk samples from an earlier 2001 USDA nationwide collection were reanalyzed. Vitamin D(3) was determined using a specifically validated method involving HPLC with UV spectroscopic detection and vitamin D(2) as an internal standard. Quality control materials were analyzed with the samples. Of the 120 milk samples procured in 2007, 49% had vitamin D(3) within 100 to 125% of 400 IU (10 μg)/qt (label value), 28% had 501 to 600 IU (12.5-15 μg)/qt, 16% had a level below the label amount, and 7% had greater than 600 IU (15 μg)/qt (>150% of label). Even though the mean vitamin D(3) content did not differ statistically between milk types, a wide range in values was found among individual samples, from nondetectable [<20 IU (0.5 μg)/qt] for one sample to almost 800 IU (20 μg)/qt, with a trend toward more samples of whole milk having greater than 150% of the labeled content. On average, vitamin D(3) in 2% milk was higher in 2007 compared with in 2001 [473 vs. 426 IU (11.8 vs. 10.6 μg)/qt].