Defensins are endogenous antimicrobial peptides. We demonstrated recently the existence of RK-1, a defensin-like peptide in rabbit kidneys. Defining the renal-defensin system is an essential step toward understanding how the kidney responds to infection. Here, using a combination of high-performance liquid chromatography, matrix-assisted laser desorption time-of-flight mass spectrometry, amino acid compositional analysis, and microsequence analysis, we show that the rabbit kidney contains a novel alpha-defensin-like peptide, RK-2. This work establishes the existence of a new subfamily of alpha-defensins in the kidney.