A large non-coding RNA, termed alpha-Fur, of similar to 1000 nt has been detected in the extreme acidophile Acidithiobacillus ferrooxidans encoded on the antisense strand to the iron-responsive master regulator fur (ferric uptake regulator) gene. A promoter for alpha-fur was predicted bioinformatically and validated using gene fusion experiments. The promoter is situated within the coding region and in the same sense as proB, potentially encoding a glutamate 5-kinase. The 3' termination site of the alpha-fur transcript was determined by 3' rapid amplification of cDNA ends to lie 7 nt downstream of the start of transcription of fur. Thus, alpha-fur is antisense to the complete coding region of fur, including its predicted ribosome-binding site. The genetic context of alpha-fur is conserved in several members of the genus Acidithiobacillus but not in all acidophiles, indicating that it is monophyletic but, not niche specific. It is hypothesized that alpha-Fur regulates the cellular level of Fur. This is the fourth example of an antisense RNA to fur, although it is the first in an extreme acidophile, and underscores the growing importance of cis-encoded non-coding RNAs as potential regulators involved in the microbial iron-responsive stimulon.