Eukaryotic ribosomes are complex structures containing 3 to 4 rRNAs and 70 to 80 distinct proteins. For cotton, and for other plants, relatively little is known about the regulation and organization of genes encoding ribosomal proteins. We began by identifying four clones encoding proteins of eukaryotic-type ribosomes from a cDNA library constructed using poly(A)(+) RNA isolated from cotton cotyledons grown without light for 72 hours. These proteins were associated with either the 40S small subunit (RS4e and RS16) or the 60S large subunit (RL41 and RL44). Multiple clones were found for all but RL41 during the initial screening. This was most likely due to small coding region of RL41 (75 bp) and the very stringent hybridization conditions. DNA sequencing of the 5' end of the clones allowed the determination of clones which encoded full length proteins. One or more full length clones were found for each ribosomal protein. Sequence comparisons of the 3' noncoding region of the clone indicated the existence of multiple copies of these clones. Each of the five cDNA's encoding RL44 contained a unique 3' noncoding sequence, indicating that at least five gene copies exist in cotton. Differences were also determined in the 3' noncoding region of clones encoding RS4e and RS16. Southern blot analysis demonstrated multiple banding patterns for each clone when hybridized to the genomic DNA of 16 cotton varieties. The deduced amino acid sequence of RS4e, RL41 and RL44 encode nuclear targeting signals which direct these ribosomal proteins into the nucleus during the synthesis of ribosomes. Cosmid libraries are presently being constructed to better elucidate the nuclear structure of these genes.