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Background Despite major progress in global vaccination coverage, immunization rates are falling, resulting in outbreaks of vaccine-preventable diseases. This study analyses content and source of the most popular tweets related to a recent case in Spain where an unvaccinated child contracted and later died from diphtheria. Understanding the characteristics of these tweets in the context of vaccination could inform efforts by health promotion professionals to increase their reach and impact. Methods We extracted tweets containing keywords related to the diphtheria case (from 1 May to 15 July 2015). We explored the prevalence of terms relating to policy and misinformation and manually coded the 194 most popular tweets (retweeted 100 or more times) with regard to source, topic, tone and sentiment. Results A total of 722 974 tweets were collected. Prevalence of terms relating to policy and misinformation increased at the onset of the case and after the death of the child. Popular tweets (194) were either pro-vaccination (58%) or neutral, with none classified as anti-vaccination. Popular topics included criticism towards anti-vaccination groups (35%) and effectiveness of immunization (22%). Popular tweets were informative (47%) or opinions (53%), which mainly expressed frustration (24%) or humour/sarcasm (23%). Popular Twitter accounts were newspaper and TV channels (15%), as well as individual journalists and authors of popular science (13.4%). Conclusions Healthcare organizations could collaborate with popular journalists or news outlets and employ authors of popular science to disseminate health information on social media, while addressing public concerns and misinformation in accessible ways. [ABSTRACT FROM AUTHOR]