Natural antisense transcripts (NATs) can interfere with the expression of complementary sense transcripts with exquisite specificity. We have previously cloned NATs of Slc34a loci (encoding Na-phosphate transporters) from fish and mouse. Here we report the cloning of a human SLC34A1-related NAT that represents an alternatively spliced PFN3 transcript (Profilin3). The transcript is predominantly expressed in testis. Phylogenetic comparison suggests two distinct mechanisms producing Slc34a-related NATs: Alternative splicing of a transcript from a protein coding downstream gene (Pfn3, human/mouse) and transcription from the bi-directional promoter (Rbpja, zebrafish). Expression analysis suggested independent regulation of the complementary Slc34a mRNAs. Analysis of randomly selected bi-directionally transcribed human/mouse loci revealed limited phylogenetic conservation and independent regulation of NATs. They were reduced on X chromosomes and clustered in regions that escape inactivation. Locus structure and expression pattern suggest a NATs-associated regulatory mechanisms in testis unrelated to the physiological role of the sense transcript encoded protein.