Objective: This study investigates effects of maternal parity, mental health and frequency of touch on infant distress expressed during routine vaccination, as well as the ability of mothers to assess infant pain. Methods: 50 mothers (19 first-time mothers, 23 stressed, 8 depressed) and their 2-month-old infants (mean age 8.1 weeks, range 7-9 weeks) were videotaped during routine immunisation for off-line-analysis. Infant-related pain behaviours were coded before, during and after needle insertion. Maternal touch was coded throughout the immunisation procedure and maternal estimates of infant pain were established immediately after the procedure. Results: Infants of first-time mothers showed significantly more pain before the needle insertion as well as during the first vaccination, compared with infants of experienced mothers. In contrast to expectation, maternal mental health status and type of touch did not influence the level of pain expressed by infants. Mothers consistently overestimated infant pain and their assessments were poorly correlated with observed infant pain behaviours. Conclusion: These findings suggest that maternal parity may influence infant pain expression before vaccination. However, all mothers overestimate their infant's pain. Further studies are required to resolve conflicting findings in this field of research, as findings may have policy implications in promoting adherence to the vaccination schedule.