In a molecular screen for polar-localized RNAs in Drosophila, we identified the mitochondrially encoded 16S large ribosomal RNA (16S RNA) as an RNA that is highly concentrated at the posterior pole of early embryos. This high posterior accumulation decreases sharply during the first hour of embryogenesis and reaches the uniform level found throughout the remainder of the embryo by the time pole cells form 1.5 hr after fertilization. At the cellular blastoderm stage the 16S RNA is uniformly distributed basal to the nuclei of all somatic cells and is present only at low levels in the pole cells and in the apical regions of the somatic cells. Transcripts produced by the 12S small rRNA gene are also concentrated in the posterior polar plasm and exhibit the same dynamic changes in distribution as the 16S RNA. In contrast, NADH dehydrogenase subunit 1 RNA, which is transcribed from the same strand of the mitochondrial genome just downstream of the 12S and 16S genes, does not exhibit a high posterior concentration but is uniformly distributed throughout early embryos. Posterior localization of 16S RNA is normal in embryos produced by mothers carrying mutations which affect posterior patterning without disrupting the polar plasm or polar granule integrity. However, posterior localization of 16S RNA is abolished in embryos produced by females carrying maternal-effect mutations that disrupt the posterior polar plasm and the polar granules. Ectopic localization of the oskar RNA to the anterior pole of the oocyte and early embryo results in anterior assembly of polar plasm and anterior budding of functional pole cells. We show that 16S and 12S RNAs are not concentrated at the anterior pole of such embryos. This leads to the conclusion that, although the 16S and 12S RNAs are concentrated in the posterior polar plasm during normal development, functional pole cells can form in the absence of high levels of these RNAs. These data argue against previous hypotheses that the 16S RNA serves an obligatory function in pole cell formation.