Background The Sexual Knowledge Picture Instrument (SKPI) is a child-friendly picture book that was developed as a diagnostic tool for sexual abuse in young children and is currently being validated. The aim of the current study was to explore the verbal and non-verbal reactions of non-abused Dutch children, aged 3-9 years, to the SKPI and to assess differences in reactions between genders and age groups. Methods and analysis Subject recruitment took place at preschools and primary schools. All children underwent a recorded video semi-structured interview with the SKPI. Interview recordings were scored by an independent rater who used scoring lists for verbal and non-verbal reactions. Non-parametric tests were used in statistical analyses. Results Of the 90 children (median age 5 years) participating in the study, most correctly named the genitals and their non-sexual functions. Only 34 showed an understanding of the picture depicting adult sexuality, with just one child describing a sexual function. Girls (n=45) gave more correct answers (median 87%) than boys (n=45, median 78%; p=0.006). Giggling was the most frequently observed non-verbal reaction, with a total of 186 instances in 54 children. Conclusion Non-abused children up to 9 years of age showed only limited knowledge of the sexual function of the genitals and hardly any insight into adult sexuality.