Introns from the Epstein-Barr virus (EBV) BART RNAs produce up to 20 micro RNAs (miRNAs) but the spliced exons of the BART RNAs have also been investigated as possible mRNAs, with the potential to express the RPMS1 and A73 proteins. Recombinant RPMS1 and A73 proteins were expressed in Escherichia coli and used to make new monoclonal antibodies that reacted specifically with artificially expressed RPMS1 and A73. These antibodies did not detect endogenous expression of A73 and RPMS1 proteins in a panel of EBV-infected cell lines representing the different known types of EBV infection. BART RNA could not be detected on Northern blots of cytoplasmic poly(A)(+) RNA from the C666.1 NPC cell line and BART RNA was found to be mainly in the nucleus of C666.1 cells, arguing against an mRNA role for BART RNAs. In contrast, some early lytic cycle EBV mRNAs were found to be expressed in C666.1 cells. Artificially expressed A73 protein was known to be able to bind to the cellular RACK1 protein and has now also been shown to be able to regulate calcium flux, presumably via RACK1. Overall, the results support the conclusion that the miRNAs are functionally important products of BART transcription in the cell lines studied because the A73 and RPMS1 proteins could not be detected in natural EBV infections. However, the possibility remains that A73 and RPMS1 might be expressed in some situations because of the clear potential relevance of their biochemical functions.