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Immunisation coverage is calculated from Australian Childhood Immunisation Register (ACIR) data using the ‘third dose assumption’. This assumes that if the third in a series of vaccine doses has been recorded on the ACIR, the previous two doses have been received, whether or not they are recorded. The objectives of this study were to validate the ‘third dose assumption’, and measure the impact of the assumption on immunisation coverage estimates at 12 months of age. A sample of children born in 1999 and assessed as fully immunised at 12 months of age by applying the ‘third dose assumption ’ were selected from the ACIR. Parents were interviewed by telephone to obtain information about vaccinations not recorded on the ACIR. Based on the survey results, the impact of the ‘third-dose assumption ’ on national coverage estimates at 12 months of age was estimated. Of 219 surveyed children assessed as up-to-date at 12 months of age only by applying the ‘third dose assumption’, 212 (96.8%) met study criteria of ‘definite ’ immunisation for all unrecorded first and second vaccine doses. Of the remaining seven, six believed all doses had been received, while one confirmed that one dose had been missed. The ‘third dose assumption ’ overestimated coverage by 0.2 per cent, based on criteria for ‘definite ’ immunisation. If the assumption were not used, immunisation coverage at 12 months of age in Australia would have been underestimated by 7 per cent. The ‘third dose assumption ’ is valid and important to use in calculating immunisation coverage from the ACIR. Although ACIR reporting and coverage levels continue to improve, under-reporting of vaccine doses due at two and four months of age persists. The ‘third dose assumption ’ may be applicable to comparable immunisation registries in other countries