As the application of nanotechnology in everyday life impacts society, it becomes critical for citizens to have a scientific basis upon which to judge their perceived hopes and fears of 'nano'. Although multiple instruments have been designed for assessing attitudinal and affective aspects of nano, surprisingly little work has focused on developing tools to evaluate the conceptual knowledge dimension of public understanding. This article reports the validation of an instrument designed to measure conceptual knowledge of nanoscience and nanotechnology. A sample of 302 participants responded to a 28-item questionnaire designed around core nano-concepts. Factor analysis revealed a single latent variable representing the construct of nano-knowledge. Cronbach's alpha was 0.91 indicating a high internal consistency of the questionnaire items. The mean test score was 15.3 out of 28 (54.5%) with item difficulty indices ranging from 0.19 to 0.89. Obtained item discrimination values indicate a high discriminatory power of the instrument. Taken together, the psychometric properties of the Nano-Knowledge Instrument (NanoKI) suggest that it is a valid and reliable tool for measuring nano-related knowledge. Preliminary qualitative observations of citizens' incorrect and correct response patterns to the questionnaire indicate potential conceptual challenges surrounding relative size of the nanoscale, random motion of nano-objects, and nanoscale interactions, although these are hypotheses that require future investigation. Application of the NanoKI could support efforts directed to an agenda for evaluating and designing science communication and education initiatives for promoting understanding of nano.