Rat aldolase B gene was confirmed to have multiple transcriptional initiation sites; one site for the major transcript and at least two sites for the minor transcripts which are located at about 30 to 60 bases upstream of the major site. Although the transcription of the minor transcripts was entirely resistant to 1 microgram/ml of alpha-amanitin, which inhibits RNA polymerase II, it was completely blocked by 1000 micrograms/ml of the drug which inhibits RNA polymerase III. The box A- and B-like sequences, which are known to exist in the internal split promoter region of class III genes, were also found to be located around the initiation sites of two minor transcripts from the rat aldolase B gene. Analyses using deletion mutants of the promoter region of the gene suggested that the synthesis of the minor transcripts depends on the box A- and B-like sequences, but does not depend on a TATA promoter element. Northern blot analysis indicated that the minor transcripts are only a few percent of the total transcripts derived from the aldolase B gene. Some of the minor transcripts analyzed were shown to have a mature size in the poly(A)+ RNA fraction. Thus, the minor transcripts of rat aldolase B gene are apparently transcribed by RNA polymerase III, and subsequently normally processed and polyadenylated. The possible function of these minor transcripts will be discussed.