The identification and characterization of a truncated goat β-casein, associated with a null β-casein allele (CSN2O′), is reported. The truncated β-casein predicted at the DNA level (NCBI Acc. No. CAB39313) but never observed at the protein level, here named β-casein O, was detected as a minor component in a goat milk sample from an autochthonous breed from southern Italy, ‘Rossa Mediterranea’, by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS). The ESI mass spectrum of the intact β-casein O determined an Mr value of 18 780 Da (calculated 18 781.5). Characterization of the amino acid sequence, performed by coupling trypsin digestion with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), RP-HPLC/ESI-MS and tandem mass spectrometry (MS/MS), demonstrated that the amino acid sequence corresponds to the 1–166 sequence of mature β-casein variant A (Acc. No. P33048), thus confirming that the protein is coded by the null allele CSN2O′, characterized by a transition (C → T) at the 373rd nucleotide of the 7th exon of the gene, which generates a premature stop codon in position 182. Copyright © 2005 John Wiley & Sons, Ltd.