Given that humans have about the same number of genes as mice and not so many more than worm, what makes us more complex? Antisense transcripts are implicated in many aspects of gene regulation. Is there a functional connection between antisense transcription and organismic complexity, that is, is antisense regulation especially prevalent in humans? We used the same robust protocol to identify antisense transcripts in humans and five other metazoan genomes (mouse, rat, chicken, fruit fly, and nematode), and found that the estimated proportions of genes involved in antisense transcription are highly sensitive to the number of transcripts included in the analysis. By controlling for transcript abundance, we find that the probability that any given transcript is putatively involved in sense-antisense regulation is no higher in humans than in other vertebrates but appears unusually high in flies and especially low in nematodes. Similarly, there is no evidence that the proportion of sense-antisense transcripts is especially higher in humans than other vertebrates in a given subset of transcript sequences such as mRNAs, coding sequences, conserved, or nonconserved transcripts. Although antisense transcription might be enriched in mammalian brains compared with nonbrain tissues, it is no more enriched in human brain than in mouse brain. Overall, therefore, while we see striking variation between multicellular animals in the abundance of antisense transcripts, there is no evidence for a link between antisense transcription and organismic complexity. More particularly, we see no evidence that humans are in any way unusual among the vertebrates in this regard. Instead, our results suggest that antisense transcription might be prevalent in almost all metazoan genomes, nematodes being an unexplained exception.