Highly parallel cDNA targeting microarrays have been established over the last years as the quasi-standard for genome wide expression profiling in pro- and eukaryotes. Protocols for the direct detection of RNA or aRNA (amplified RNA) are currently emerging. This allows to circumvent the bias introduced by enzymatic target molecule preparation. To systematically evaluate the extent of non-specific target binding on oligonucleotide microarrays designed for total RNA expression profiling, a model system of 70-mer probes targeting genes involved in magnetosome formation (mam genes) of the bacterium Magnetospirillum gryphiswaldense was established utilizing wild-type strain MSR-1 and an isogenic deletion mutant MSR-1B that lacks all mam genes. An optimized protocol for the direct chemical labelling of total cellular RNAs was used. A linear correlation between the amount of applied RNA and the mean global background intensity was found which enables a simple and unbiased way of normalizing the data. The results obtained with the mam deletion mutant MSR-1B revealed a significant number of false positive signals, even under optimal hybridization conditions. This indicates a high degree of non-specific binding in microarray experiments when using longer oligo- or polynucleotides and RNA as target molecule. Comparative microarray analysis of an MSR-1B culture and two MSR-1 wild-type cultures grown under different conditions was done via a three-colour hybridization assay. The additional information provided by the MSR-1B transcriptome revealed differential gene expression in the two MSR-1 cultures, which was otherwise undetectable.