A locus responsible for autosomal recessive spinal muscular atrophy (SMA) on chromosome 5q11.2-q13.3 has been mapped to a critical interval delimited by markers D5S435 and D5S557. By a modification of the Vectorette-(GT)n method, we have isolated three polymorphic CA repeats from two YACs of the SMA region. Two of them (D5S1417 and D5S1416) map within the SMA critical region, and the other (D5S1415) is centromeric to D5S435. Linkage analysis in Spanish SMA families with eleven markers showed that in our families the disease is linked to this region and confirmed that the novel markers are tightly linked to the SMA locus. The most likely order of markers was 5cen-(D5S63/D5S1356)-(D5S125/D5S465)- (D5S435/D5S1417/D5S1416/D5S557)-D5S610- D5S112-D5S127-5qter, with odds against alternative orders > 1,000:1. Genetic distances are in agreement with those previously published. However, the recombination fraction between D5S610 and D5S112 is remarkably greater than expected from the physical distance, suggesting a hot spot for recombination in this region. Our results from haplotype and multipoint analyses show that the SMA locus must lie between D5S465 and D5S112, and lend further support to the current location of the SMA locus.