The processing and ribonucleoprotein assembly of U4 small nuclear RNA has been investigated in HeLa cells. After a 45-min pulse label with [3H]uridine, a set of apparently cytoplasmic RNAs was observed migrating just behind the gel electrophoretic position of mature U4 RNA. These molecules were estimated to be one to at least seven nucleotides longer than mature U4 RNA. They reacted with Sm autoimmune patient sera and a monoclonal Sm antibody, indicating their association with proteins characteristic of small nuclear ribonucleoprotein complexes. The same set of RNAs was identified by hybrid selection of pulse-labeled RNA with cloned U4 DNA, confirming that these are U4 RNA sequences. No larger nuclear precursors of these RNAs were detected. Pulse-chase experiments revealed a progressive decrease in the radioactivity of the U4 precursor RNAs coincident with an accumulation of labeled mature U4 RNA, confirming a precursor-product relationship.