A comparison of isogenic RNase III+ and RNase III- strains of Escherichia coli shows that although both synthesize precursor and mature 16 S and 23 S ribosomal RNAs, the transient rRNA species of the RNase III- strain differ from those of the RNase III+ strain. The RNase III+ strain synthesizes p16 and p23 rRNA, whereas the RNase III- strain produces unstable 17 S, 18 S, "p23," 25 S and 30 S RNA molecules. The 30 S RNA, which is a primary transcript of the ribosomal RNA gene cluster, does not contribute significantly to any of the smaller RNAs, nor is m23 rRNA derived from 25 S but rather from "p23" RNA. Mature 16 S rRNA is derived from both 18 S and 17 S RNA, and 17 S RNA can be derived from 18 S. Additionally, an unstable RNA species about 300 bases long is missing in the RNase III- strain and another species which seems to be about 50 bases larger appears. Processing of the primary ribosomal RNA transcript in RNase III- strains of Escherichia coli is accomplished during its transcription by two independent pathways which are not so utilized in RNase III+ strains. One pathway yields 18 S and precursor 23 S RNAs which are processed to mature rRNAs; the second pathway yields 25 S RNA and perhaps 16 S rRNA. The second pathway, unlike the first, is inhibited by chloramphenicol treatment. At slow rates of ribosomal RNA synthesis, the nascent transcript is processed preferentially by the first pathway. We suggest that in the absence of RNase III, which is involved in the primary processing of rRNA in E. coli, other enzymes involved in primary and secondary processing of rRNA in RNase III+ cells can recognize their sites on the nascent rRNA transcript and accomplish the primary processing.