Innate immunity plays an important role in preventing (barrier function) or combating infection (effector function). An important humoral component of innate immunity is formed by natural antibodies (NAb). The objectives of this study were to determine presence, variation among cows and repeatability within cows over time of total NAb titers directed to the pathogen-associated molecular patterns lipopolysaccharide, lipoteichoic acid (LTA) and peptidoglycan, and titers of NAb directed to the glycoprotein keyhole limpet hemocyanin in milk and plasma of individual cows. Furthermore in milk the antibody isotypes IgG1, IgG2, IgM and IgA binding LTA were analyzed. Ten milk and blood samples were obtained from each of 20 clinically healthy dairy cows from first to seventh parity during a period of 3 weeks. Total NAb binding lipopolysaccharide, LTA, peptidoglycan, and keyhole limpet hemocyanin were detected in milk and plasma, with titers considerably higher in plasma than in milk. Total NAb titers showed significant variation among cows, and repeatability within cows over time (ranging from 0.60 to 0.93). Titers of NAb in milk and plasma were positively correlated (correlation ranging from 0.69 to 0.91). Natural antibodies in milk binding LTA were of all 4 isotypes tested, although IgG2 was on average only present at low titers. All 4 isotypes in milk binding LTA also showed variation among cows, and repeatability within cows over time (ranging from 0.84 to 0.92). We conclude that NAb can be measured in a consistent and repeatable manner in bovine milk and blood plasma.