The Particulate Nature of Matter and Chemical Bonding Diagnostic Instrument (Othman J., Treagust D. F. and Chandrasegaran A. L., (2008), "Int. J. Sci. Educ.," 30(11), 1531-1550) is used to investigate college students' understanding of two chemistry concepts: particulate nature of matter and chemical bonding. The instrument, originally developed for secondary school students, is a two-tier diagnostic test. In this study, the instrument was given as a paper and pencil test to college students enrolled in General Chemistry I during the second week of class in Spring 2010. General Chemistry I students were divided into three groups for analysis: (A) with preparatory chemistry, (B) repeating general chemistry (without preparatory chemistry); and (C) first time in general chemistry (without preparatory chemistry); Groups B and C are expected to have taken at least one year of secondary school chemistry. However, Group A had little or no exposure to high school chemistry. Regardless of their pathway into the course, students in General Chemistry I are expected to have a fair understanding of the two concepts. Furthermore, students' responses were used to think about the role of a preparatory chemistry course in promoting deep understanding of chemistry concepts. An ANCOVA analysis was performed. Preparatory Chemistry was found to have a statistically significant but small effect on students' scores after controlling for prior math achievement. Confirmatory factor analysis and reliability studies were performed and are presented here. Analysis also revealed alternative conceptions. (Contains 6 tables.)