A large-scale functional genomics project was initiated to study the function of chromatin-related genes in maize (Zea mays). Transgenic lines containing short gene segments in inverted repeat orientation designed to reduce expression of target genes by RNA interference (RNAi) were isolated, propagated, and analyzed in a variety of assays. Analysis of the selectable marker expression over multiple generations revealed that most transgenes were transmitted faithfully, whereas some displayed reduced transmission or transgene silencing. A range of target-gene silencing efficiencies, from nondetectable silencing to nearly complete silencing, was revealed by semiquantitative reverse transcription-PCR analysis of transcript abundance for the target gene. In some cases, the RNAi construct was able to cause a reduction in the steady-state RNA levels of not only the target gene, but also another closely related gene. Correlation of silencing efficiency with expression level of the target gene and sequence features of the inverted repeat did not reveal any factors capable of predicting the silencing success of a particular RNAi-inducing construct. The frequencies of success of this large-scale project in maize, together with parameters for optimization at various steps, should serve as a useful framework for designing future RNAi-based functional genomics projects in crop plants.