Most PRKAR1A tumorigenic mutations lead to nonsense mRNA that is decayed; tumor formation has been associated with an increase in type II protein kinase A (PKA) subunits. The IVS6+1G>T PRKAR1A mutation leads to a protein lacking exon 6 sequences [R1 alpha Delta 184-236 (R1 alpha Delta 6)]. We compared in vitro R1 alpha Delta 6 with wild-type (wt) R1 alpha. We assessed PKA activity and subunit expression, phosphorylation of target molecules, and properties of wt-R1 alpha and mutant (mt) R1 alpha; we observed by confocal microscopy R1 alpha tagged with green fluorescent protein and its interactions with Cerulean-tagged catalytic subunit (C alpha). Introduction of the R1 alpha Delta 6 led to aberrant cellular morphology and higher PKA activity but no increase in type II PKA subunits. There was diffuse, cytoplasmic localization of R1 alpha protein in wt-R1 alpha- and R1 alpha Delta 6-transfected cells but the former also exhibited discrete aggregates of R1 alpha that bound C alpha; these were absent in R1 alpha Delta 6-transfected cells and did not bind C alpha at baseline or in response to cyclic AMP. Other changes induced by R1 alpha Delta 6 included decreased nuclear C alpha. We conclude that R1 alpha Delta 6 leads to increased PKA activity through the mt-R1 alpha decreased binding to C alpha and does not involve changes in other PKA subunits, suggesting that a switch to type II PKA activity is not necessary for increased kinase activity or tumorigenesis.