In a recently published study, Anna Krichevsky and colleagues raise the important question ofwhether results of in vitro extracellular RNA (exRNA) studies, including extracellular vesicle (EV)investigations, are confounded by the presence of RNA in cell culture medium components suchas foetal bovine serum (FBS). The answer, according to their data, is a resounding“yes”. Even afterlengthy ultracentrifugation to remove bovine EVs from FBS, the majority of exRNA in FBSremained. Although technical factors may affect the degree of depletion, residual EVs andexRNA in FBS could influence the conclusions of in vitro studies: certainly, for secreted RNA,and possibly also for cell-associated RNA. In this commentary, we critically examine some of theliterature in this field, including a recent study from some of the authors of this piece, in light ofthe Wei et al. study and explore how cell culture-derived RNAs may affect what we think we knowabout EV RNAs. These findings hold particular consequence as the field moves towards a deeperunderstanding of EV–RNA associations and potential functions.