Several distinct domains of U4 small nuclear (sn)RNA interact with other components of the spliceosome and are essential for pre-mRNA splicing. We have previously shown that single point mutations (Hu et al., 1995) in the central domain of the yeast U4 snRNA do not effect cell growth. In contrast to our results, this region has been reported to possess metal-binding or catalytic activity in vitro (Yang et al., 1994). In order to test if large mutations in the central domain effect cell growth and/or in vivo splicing, we have carried out further mutational analyses of this domain. A deletion of nucleotides from 62 to 88, including all those corresponding to the invariable positions of the hypothetical hammerhead, does not affect cell growth at/or above 25 degrees C. In addition, the central region (nucleotides 69-88) can be replaced by two copies of a 38-nt spacer inserted in the same orientation without any effect on cell growth or nuclear RNA splicing. These results suggest that the central domain of U4 snRNA functions primarily to separate the 5'- and 3'- domains of the snRNA by an optimal distance.