The recycling of ribosomes at stop codons for use in further rounds of translation is critical for efficient protein synthesis. Removal of the 60S subunit is catalyzed by the ATPase Rli1 (ABCE1) while removal of the 40S is thought to require Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR). However, it remains unclear how these Tma proteins cause 40S removal and control reinitiation of downstream translation. Here we developed an enhanced 40S ribosome footprinting strategy to directly observe intermediate steps of ribosome recycling in cells. Deletion of the genes encoding these Tma proteins resulted in broad accumulation of unrecycled 40S subunits at stop codons, directly establishing their role in 40S recycling. Furthermore, the Tma20/Tma22 heterodimer was responsible for a majority of 40S recycling events while Tma64 played a minor role. Introduction of an autism-associated mutation into TMA22 resulted in a loss of 40S recycling activity, linking ribosome recycling and neurological disease. Overall design: 40S ribosome profiling of 12 biological samples with 11 replicates. 80S ribosome profiling of 4 biological samples with one replicate. Deletion strains and mutations affect either translation initiation (rpl11b) or ribosome recycling (tma64 /tma20 , tma64 /tma22 , tma20 , tma22 , tma64 , tma22 YCplac33, tma22 YCplac33-TMA22, tma22 YCplac33-DENR, tma22 YCplac33-tma22-C11Y, tma22 YCplac33-tma22-A105L) + WT. Please note that wig files composed of combination of two biological replicates (e.g. 26-45end5_DY134Ffx182Ffx_.wig) are linked to the sample with the lower number (e.g. DY134Ffx sample for 26-45end5_DY134Ffx182Ffx_.wig) as indicated in the corresponding sample description field.