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Highly pathogenic avian influenza (HPAI) H5N1 haemagglutinin clade 2.3.4.4b was detected in the USA in 2021. These HPAI viruses caused mortality events in poultry, wild birds and wild mammals. On 25 March 2024, HPAI H5N1 clade 2.3.4.4b was confirmed in a dairy cow in Texas in response to a multistate investigation into milk production losses¹. More than 200 positive herds were identified in 14 US states. The case description included reduced feed intake and rumen motility in lactating cows, decreased milk production and thick yellow milk^2,3. The diagnostic investigation revealed viral RNA in milk and alveolar epithelial degeneration and necrosis and positive immunoreactivity of glandular epithelium in mammary tissue. A single transmission event, probably from birds, was followed by limited local transmission and onward horizontal transmission of H5N1 clade 2.3.4.4b genotype B3.13 (ref. ⁴). Here we sought to experimentally reproduce infection with genotype B3.13 in Holstein yearling heifers and lactating cows. Heifers were inoculated by an aerosol respiratory route and cows by an intramammary route. Clinical disease was mild in heifers, but infection was confirmed by virus detection, lesions and seroconversion. Clinical disease in lactating cows included decreased rumen motility, changes to milk appearance and production losses. Infection was confirmed by high levels of viral RNA detected in milk, virus isolation, lesions in mammary tissue and seroconversion. This study provides the foundation to investigate additional routes of infection, pathogenesis, transmission and intervention strategies.