BACKGROUND: Natural antisense transcripts (NATs) are coding or non-coding RNAs with sequence complementarity to other transcripts (sense transcripts). These RNAs could potentially regulate the expression of their sense partner(s) at either the transcriptional or post-transcriptional level. Experimental and computational methods have demonstrated the widespread occurrence of NATs in eukaryotes. However, most previous studies only focused on cis-NATs with little attention being paid to NATs that originate in trans. RESULTS: We have performed a genome-wide screen of trans-NATs in Arabidopsis thaliana and identified 1,320 putative trans-NAT pairs. An RNA annealing program predicted that most trans-NATs could form extended double-stranded RNA duplexes with their sense partners. Among trans-NATs with available expression data, more than 85% were found in the same tissue as their sense partners; of these, 67% were found in the same cell as their sense partners at comparable expression levels. For about 60% of Arabidopsis trans-NATs, orthologs of at least one transcript of the pair also had trans-NAT partners in either Populus trichocarpa or Oryza sativa. The observation that 430 transcripts had both putative cis- and trans-NATs implicates multiple regulations by antisense transcripts. The potential roles of trans-NATs in inducing post-transcriptional gene silencing and in regulating alternative splicing were also examined. CONCLUSION: The Arabidopsis transcriptome contains a fairly large number of trans-NATs, whose possible functions include silencing of the corresponding sense transcripts or altering their splicing patterns. The interlaced relationships observed in some cis- and trans-NAT pairs suggest that antisense transcripts could be involved in complex regulatory networks in eukaryotes.