The aim of this study was to detect new polymorphisms in the bovine ß-casein (ß-CN) gene and to evaluate association of (new) ß-CN protein variants with milk production traits and milk protein composition. Screening of the ß-CN gene in genomic DNA from 72 Holstein Friesian (HF) bulls resulted in detection of 19 polymorphisms and revealed the presence of ß-CN protein variant I in the Dutch HF population. Studies of association of ß-CN protein variants with milk composition usually do not discriminate protein variant I from variant A2. Association of ß-CN protein variants with milk composition was studied in 1857 first-lactation HF cows and showed that associations of protein variants A2 and I were quite different for several traits. ß-CN protein variant I was significantly associated with protein percentage and protein yield, and with as1-casein (as1-CN), as2-casein (as2-CN), ¿-casein (¿-CN), a-lactalbumin (a-LA), ß-lactoglobulin (ß-LG), casein index and casein yield. Inferring ß-¿-CN haplotypes showed that ß-CN protein variant I occurred only with ¿-CN variant B. Consequently, associations of ß-¿-CN haplotype IB with protein percentage, ¿-CN, a-LA, ß-LG and casein index are likely resulting from associations of ¿-CN protein variant B, while associations of ß-¿-CN haplotype IB with as1-CN and as2-CN seem to be resulting from associations of ß-CN variant I.